Synthesis of Prazole Compounds

ABSTRACT

The present disclosure relates to non-naturally occurring monooxygenase polypeptides useful for preparing prazole compounds, polynucleotides encoding the polypeptides, and methods of using the polypeptides.

1. TECHNICAL FIELD

The present disclosure relates to biocatalysts and methods of using thebiocatalysts.

2. REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM

The Sequence Listing is concurrently submitted herewith thespecification as an ASCII formatted text file via EFS-Web with a filename of CX2-036WO1_S′T25.txt with a creation date of Dec. 7, 2010, and asize of 955175 bytes. The Sequence Listing filed via EFS-Web is part ofthe specification and is hereby incorporated in its entirety byreference herein.

3. BACKGROUND

Esomeprazole is a proton pump inhibitor prescribed for the treatment ofdyspepsia, peptic ulcer disease (PUD), gastroesophageal reflux disease(GORD/GERD) and Zollinger-Ellison syndrome. Esomeprazole is theS-enantiomer of omeprazole, which is a racemic mixture of the S and Risomers. Esomeprazole (also referred to herein as“(S)-5-methoxy-2-((4-methoxy-3,5-dimethylpyridin-2-yl)methylsulfinyl)-1H-benzo[d]imidazole”or “compound (2b)”) has the following structure:

Esomeprazole and its corresponding R-isomer is a prodrug that isconverted to the active form in acidic environments. It is activated bya proton catalyzed process to form a sulphenamide, which interacts withthe sulfhydryl groups of cysteine residues in the extracellular domainof H⁺K⁺-ATPases, thereby inhibiting its activity. The efficacy of theS-enantiomer is indicated as being greater than the racemic omeprazole.

Esomeprazole is typically synthesized by chemical asymmetric oxidationof sulfides to sulfoxides, i.e., a Kagan-Sharpless type oxidation, asdescribed in Cotton et al., 2000, Tetrahedron: Asymmetry 11:3819. Theprocess results in esomeprazole in about 94% enantiomeric excess. Theenantiopurity of esomeprazole preparations can be increasedsubstantially by preparing a magnesium salt followed by crystallization.Different salts and hydrates of esomeprazole have also been described.For example, WO 00/44744 discloses the potassium salt of esomeprazole.U.S. Pat. No. 6,162,816 discloses crystalline form A and lesscrystalline form B of neutral esomeprazole, prepared by arecrystallization from ethyl acetate, methylene chloride or toluene.U.S. Pat. No. 6,369,085 discloses esomeprazole magnesium trihydrateprepared from the corresponding potassium salt, precipitated withacetone, and treated with water.

However, it is desirable to increase the efficiency of manufacture aswell as reducing the number of processing steps for forming esomeprazolepreparations of high enantiopurity. It is also desirable to identifyprocesses that are applicable to preparation of other prazole compoundsin addition to esomeprazole.

4. SUMMARY

The present disclosure provides polypeptides, polynucleotides encodingthe polypeptides and methods of using the polypeptides for thebiocatalytic sulfoxidation of prazole compounds, in particular theconversion of the sulfide substrate of5-methoxy-2-((4-methoxy-3,5-dimethylpyridin-2-yl)methylthio)-1H-benzo[d]imidazole(also referred to herein as “compound (1)”) to the (R)- and(S)-omeprazole product of5-methoxy-2-((4-methoxy-3,5-dimethylpyridin-2-yl)methylsulfinyl)-1H-benzo[d]imidazole(also referred to herein as “compound (2)”).

While naturally occurring cyclohexanone monooxygenase (CHMO)polypeptides do not efficiently convert compound (1) to compound (2),the non-naturally occurring monooxygenase polypeptides (also referred toherein as “engineered CHMO polypeptides”) of the present disclosure havebeen designed to efficiently carryout the conversion. Moreover, in someembodiments, the non-naturally occurring monooxygenase polypeptides ofthe disclosure can carry out the reaction stereoselectively to the formthe S-isomer,(S)-5-methoxy-2-((4-methoxy-3,5-dimethylpyridin-2-yl)methylsulfinyl)-1H-benzo[d]imidazole(also referred to herein as “compound (2b)”) in enantiomeric excess overthe R-isomer, or in some embodiments, to form(R)-5-methoxy-2-((4-methoxy-3,5-dimethylpyridin-2-yl)methylsulfinyl)-1H-benzo[d]imidazole(also referred to herein as “compound (2a)”) in enantiomeric excess overthe S-isomer. In some embodiments, the monooxygenase polypeptides canproduce compound (2b) as a substantially enantiomerically purepreparation.

In some embodiments, the polypeptide is capable of converting compound(1) to compound (2a) in enantiomeric excess. In some embodiments, theR-enantioselective monooxygenase polypeptide comprises an amino acidsequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to a referencesequence selected from the group consisting of SEQ ID NO: 4, 6, 160,162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188,190, 192, 194, 196, 198, 200, 202, 204, 206, and 208. In particular, thereference sequence is SEQ ID NO: 6, 166, 170, 174, 190, 192, 196, 200,204, or 206.

In some embodiments, the polypeptide is capable of converting compound(1) to compound (2b) in enantiomeric excess. In some embodiments, theS-enantioselective monooxygenase polypeptide comprises an amino acidsequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to a referencesequence selected from the group consisting of SEQ ID NO: 8, 10, 22, 52,76, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114,116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142,144, 146, 148, 150, 152, 154, 156, 158, 210, 212, 214, 216, 218, 220,222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248,250, 252, 254, 256, 258, 260, 262, 264, and 266. In particular, thereference sequence is SEQ ID NO: 116, 124, 130, 138, or 158.

Additionally, the present disclosure provides a monooxygenasepolypeptide capable of converting compound (1) to compound (2) at a ratethat is improved over the naturally occurring monooxygenase of SEQ IDNO:2. In some embodiments, the non-naturally occurring monooxygenasepolypeptide of the disclosure are capable of converting compound (1) tocompound (2) at a rate that is greater than 1.5 fold the rate of SEQ IDNO:2. In some embodiments, the polypeptide is capable of convertingcompound (1) to compound (2) at a rate that is greater than 1.5 fold thepolypeptide of SEQ ID NO:2 and comprises an amino acid sequence that isat least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or more identical to a reference sequence selectedfrom SEQ ID NO: 4, 6, 8, 10, 22, 52, 76, 86, 88, 90, 92, 94, 96, 98,100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126,128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154,156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182,184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210,212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238,240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, or 266.In particular, the reference sequence is SEQ ID NO: 116, 124, 130, 138,or 158.

The polypeptide capable of converting compound (1) to compound (2) withimproved enantioselectivity and/or enzymatic activity, having beenderived from the naturally occurring cyclohexanone monooxygenase ofAcinetobacter NCIMB9871, comprises an amino acid sequence that has oneor more residue differences as compared to the sequence of SEQ ID NO:2.The residue differences can occur at residue positions identified asbeing associated with desirable changes in enzyme activity,enantioselectivity, sulfone-byproduct formation, thermostability,solvent stability, expression, or various combinations thereof.Accordingly, in some embodiments, the polypeptides of the disclosure canhave one or more residue differences as compared to SEQ ID NO:2 at thefollowing residue positions: X3; X14; X34; X43; X71; X111; X141; X149,X174; X209; X240; X246; X248; X288; X307; X326; X383; X386; X388; X390;X400; X415; X426; X432; X433; X435; X438; X448; X449; X481; X488; X489;X490; X499; X505; X516; X526; X537; and X540. In some embodiments, themonooxygenase amino acid sequence has at least two or more, at leastthree or more, or at least four or more residue differences at theresidue positions above as compared to the reference sequence of SEQ IDNO:2. Amino acid residues that can be present at these positions areprovided in the detailed descriptions herein.

In some embodiments, the polypeptide comprises an amino acid sequencehaving one or more residue differences as compared to SEQ ID NO:2 atresidue positions associated with changes in enantioselectivity, whichpositions are selected from the following: X246; X248; X326; X386; X432;X433; X435; X438; and X448. In some embodiments, the monooxygenase hasat least two or more, at least three or more, or at least four or moreresidue differences at these residues positions as compared to thereference sequence of SEQ ID NO:2.

In some embodiments, the non-naturally occurring monooxygenasepolypeptide comprises an amino acid sequence having one or more residuedifferences as compared to SEQ ID NO:2 at residue positions associatedwith decreases in sulfone-byproduct formation, where thesulfone-byproduct has the following structure of compound (3):

Residue positions associated with a decrease in the amount ofsulfone-byproduct of compound (3) formed in the biocatalytic process canbe selected from the following: X246, X248, X277, and X438.

In some embodiments, the polypeptide comprises an amino acid sequencehaving one or more residue differences as compared to SEQ ID NO:2 atresidue positions associated with increases in thermostability and/orsolvent stability, which positions are selected from the following: X43,X71, X111, X149, X174, X307; X341, X368, X388, X390, X400, X449, X481,and X488.

In some embodiments, the polypeptide comprises an amino acid sequencehaving residue differences as compared to SEQ ID NO:2 at residuepositions associated with increases in protein expression, whichposition includes X3.

As will be apparent to the skilled artisan, various combinations ofresidue differences as compared to SEQ ID NO:2 at residue positionsassociated with desirable changes in enzymatic activity,enantioselectivity, sulfone-byproduct formation, thermostability,solvent stability and expression can be used to form the polypeptides ofthe present disclosure.

In some embodiments, the polypeptide capable of converting compound (1)to compound (2a) in enantiomeric excess (R-enantioselective) comprisesan amino acid sequence which comprises at least two or more of thefollowing features: residue at position corresponding to X432 is analiphatic amino acid residue; residue at position corresponding to X433is a non-polar amino acid residue; residue at position corresponding toX435 is a hydroxyl-containing amino acid residue; and residue atposition corresponding to X490 is a basic amino acid residue. In someembodiments, the R-enantioselective monooxygenases have at least threeof the above features, or at least all of the above features.

In some embodiments, the polypeptide capable of converting compound (1)to compound (2b) in enantiomeric excess (S-selective) comprises an aminoacid sequence in which at least residue at position corresponding toX326 is a cysteine (C). In some embodiments, the polypeptide capable ofconverting compound (1) to compound (2b) in enantiomeric excesscomprises an amino acid sequence in which at least residue at positioncorresponding to X386 is a hydroxyl-containing amino acid residue,particularly a S.

In some embodiments, the polypeptide capable of converting compound (1)to compound (2b) in enantiomeric excess comprises an amino acid sequencewhich includes at least the following features: residue at positioncorresponding to X326 is a cysteine (C) and residue at positioncorresponding to X386 is a hydroxyl-containing amino acid residue,particularly a S.

In some embodiments, the S-enantioselective monooxygenase polypeptidescomprises an amino acid sequence having, in addition to residuedifferences associated with S-enantioselectivity above, at least one ormore residue differences at residue positions associated with increasedenzyme activity for the pyrmetazole substrate and/or increasedS-enantioselectivity, particularly at residue positions X432, X433,X435, X438, X448, and X490, more particularly at least one or moreresidue differences at residue positions X432, X433, X435, and X490.

Thus, in some embodiments, the polypeptide capable of convertingcompound (1) to compound (2b) in enantiomeric excess comprises an aminoacid sequence which includes at least the following features: residue atposition corresponding to X326 is a cysteine (C); residue at positioncorresponding to X386 is a hydroxyl-containing amino acid residue; andresidue at position corresponding to X432 is an aliphatic amino acidresidue.

In some embodiments, the polypeptide capable of converting compound (1)to compound (2b) in enantiomeric excess comprises an amino acid sequencewhich includes at least the following features: residue at positioncorresponding to X326 is a cysteine (C); residue at positioncorresponding to X386 is a hydroxyl-containing amino acid residue;residue at position corresponding to X432 is an aliphatic amino acidresidue; residue at position corresponding to X433 is an aliphatic aminoacid residue, residue at position corresponding to X435 is ahydroxyl-containing amino acid residue; and residue at positioncorresponding to X490 is a basic amino acid residue.

In some embodiments, the polypeptide capable of converting compound (1)to compound (2b) in enantiomeric excess comprises an amino acid sequencewhich includes at least the following features: residue at positioncorresponding to X326 is C; residue at position corresponding to X386 isS; residue at position corresponding to X432 is A, or L; residue atposition corresponding to X433 is A, L, or V; residue at positioncorresponding to X435 is S; and residue at position corresponding toX490 is R.

In some embodiments, the polypeptide capable of converting compound (1)to compound (2b) in enantiomeric excess comprises an amino acid sequencewhich includes at least the following features: residue at positioncorresponding to X34 is K; residue at position corresponding to X209 isP; residue at position corresponding to X240 is F or K; residue atposition corresponding to X288 is I; residue at position correspondingto X326 is C; residue at position corresponding to X386 is S; residue atposition corresponding to X415 is A; residue at position correspondingto X432 is L; residue at position corresponding to X433 is A; residue atposition corresponding to X435 is S; residue at position correspondingto X438 is I; residue at position corresponding to X448 is V; residue atposition corresponding to X490 is R; residue at position correspondingto X516 is V; and residue at position corresponding to X537 is T.

In some embodiments, the polypeptide capable of converting compound (1)to compound (2b) in enantiomeric excess comprises an amino acid sequencewhich includes at least the following features: residue at positioncorresponding to X14 is A; residue at position corresponding to X34 isK; residue at position corresponding to X141 is V; residue at positioncorresponding to X209 is P; residue at position corresponding to X240 isF or K; residue at position corresponding to X288 is I; residue atposition corresponding to X326 is C; residue at position correspondingto X386 is S; residue at position corresponding to X415 is A; residue atposition corresponding to X426 is F; residue at position correspondingto X432 is L; residue at position corresponding to X433 is A; residue atposition corresponding to X435 is S; residue at position correspondingto X438 is I; residue at position corresponding to X448 is V; residue atposition corresponding to X489 is C; residue at position correspondingto X490 is R; residue at position corresponding to X516 is V; andresidue at position corresponding to X537 is T.

In some embodiments, the polypeptide capable of converting compound (1)to compound (2b) in enantiomeric excess comprises an amino acid sequencewhich includes at least the following features: residue at positioncorresponding to X14 is A; residue at position corresponding to X34 isK; residue at position corresponding to X111 is T; residue at positioncorresponding to X141 is V; residue at position corresponding to X209 isP; residue at position corresponding to X240 is F or K; residue atposition corresponding to X246 is Y; residue at position correspondingto X288 is I; residue at position corresponding to X307 is C or R;residue at position corresponding to X326 is C; residue at positioncorresponding to X386 is S; residue at position corresponding to X388 isK; residue at position corresponding to X415 is A; residue at positioncorresponding to X426 is F; residue at position corresponding to X432 isL; residue at position corresponding to X433 is A; residue at positioncorresponding to X435 is S; residue at position corresponding to X438 isI; residue at position corresponding to X448 is V; residue at positioncorresponding to X481 is K; residue at position corresponding to X489 isC; residue at position corresponding to X490 is R; residue at positioncorresponding to X505 is L; residue at position corresponding to X516 isV; and residue at position corresponding to X537 is T.

In some embodiments, the polypeptide capable of converting compound (1)to compound (2b) in enantiomeric excess comprises an amino acid sequencewhich includes at least the following features: residue at positioncorresponding to X14 is A; residue at position corresponding to X34 isK; residue at position corresponding to X43 is G; residue at positioncorresponding to X71 is M; residue at position corresponding to X111 isT; residue at position corresponding to X141 is V; residue at positioncorresponding to X149 is W; residue at position corresponding to X209 isP; residue at position corresponding to X240 is F or K; residue atposition corresponding to X246 is Y; residue at position correspondingto X248 is I or V; residue at position corresponding to X277 is M;residue at position corresponding to X288 is I; residue at positioncorresponding to X307 is C or R; residue at position corresponding toX326 is C; residue at position corresponding to X386 is S; residue atposition corresponding to X388 is K; residue at position correspondingto X390 is I; residue at position corresponding to X415 is A; residue atposition corresponding to X426 is F; residue at position correspondingto X432 is L; residue at position corresponding to X433 is A; residue atposition corresponding to X435 is S; residue at position correspondingto X438 is I; residue at position corresponding to X448 is V; residue atposition corresponding to X481 is K; residue at position correspondingto X489 is C; residue at position corresponding to X490 is R; residue atposition corresponding to X499 is G, L, or R; residue at positioncorresponding to X505 is L; residue at position corresponding to X516 isV; residue at position corresponding to X526 is V; residue at positioncorresponding to X537 is T; and residue at position corresponding toX540 is Q.

In some embodiments, the polypeptide capable of converting compound (1)to compound (2b) in enantiomeric excess comprises an amino acid sequencewhich includes at least the following features: residue at positioncorresponding to X14 is A; residue at position corresponding to X34 isK; residue at position corresponding to X43 is G; residue at positioncorresponding to X71 is M; residue at position corresponding to X111 isT; residue at position corresponding to X141 is V; residue at positioncorresponding to X149 is W; residue at position corresponding to X174 isI; residue at position corresponding to X209 is P; residue at positioncorresponding to X240 is F or K; residue at position corresponding toX246 is Y; residue at position corresponding to X248 is I or V; residueat position corresponding to X277 is M; residue at positioncorresponding to X288 is I; residue at position corresponding to X307 isC or R; residue at position corresponding to X326 is C; residue atposition corresponding to X341 is E; residue at position correspondingto X383 is G; residue at position corresponding to X386 is S; residue atposition corresponding to X388 is K; residue at position correspondingto X390 is I; residue at position corresponding to X400 is I; residue atposition corresponding to X415 is A; residue at position correspondingto X426 is F; residue at position corresponding to X432 is L; residue atposition corresponding to X433 is A; residue at position correspondingto X435 is S; residue at position corresponding to X438 is I; residue atposition corresponding to X448 is V; residue at position correspondingto X449 is F; residue at position corresponding to X481 is K; residue atposition corresponding to X488 is K; residue at position correspondingto X489 is C; residue at position corresponding to X490 is R; residue atposition corresponding to X499 is G, L, or R; residue at positioncorresponding to X505 is L; residue at position corresponding to X516 isV; residue at position corresponding to X526 is V; residue at positioncorresponding to X537 is T; and residue at position corresponding toX540 is Q.

In addition to the residue positions specified above, various otherresidue differences relative to SEQ ID NO:2 can be present at otherresidue positions in the non-naturally occurring monooxygenasepolypeptides disclosed herein. These can be conservative ornon-conservative differences, including conservative substitutions andnon-conservative substitutions. Guidance on these other residuepositions and the choices of amino acid residues at these otherspecified positions is provided in the detailed description.

In another aspect, provided herein are polynucleotides encoding themonooxygenase polypeptides, expression vectors comprising thepolynucleotides, and host cells capable of expressing the polypeptides.

In a further aspect, provided herein are processes of using thepolypeptides of the disclosure for converting a substrate compound ofstructural formula (I) to the product compound of formula (II):

wherein,

Ar¹ is an optionally substituted aryl or heteroaryl ring;

R is H, a lower alkyl, a heteroalkyl, or forms a 5 to 8 memberedcycloalkyl, heteroalkyl, aryl or heteroaryl fused ring with a ringcarbon of Ar¹; and

Ar² is an optionally substituted cycloalkyl, heterocycloalkyl, aryl, orheteroaryl ring fused to the imidazole ring.

Generally, the process comprises contacting the compound of formula (I)with an engineered monooxygenase polypeptide described herein inpresence of a cofactor under suitable reaction conditions for convertingthe substrate to the product compound of formula (II). The suitablereaction conditions typically include a source of molecular oxygen O₂,and the cofactor is NADPH or NADH.

processes of using the polypeptides of the disclosure can be used in thepreparation of omeprazole analog compounds of structural formula (II) inenantiomeric excess, wherein the compounds of structural formula (II)are selected from: (R) or (S)-lansoprazole, (R) or (3)-tenatoprazole,(R) or (S)-rabeprazole, (R) or (S)-pantoprazole, (R) or (S)-ilaprazole,(R) or (S)-leminoprazole, (R) or (S)-saviprazole, and (R) or(S)-TY-11345.

In some embodiments, Ar¹ is an optionally substituted phenyl or pyridyl.In some embodiments, Ar² is an optionally substituted thienyl, phenyl orpyridyl. In some embodiments, the compound of formula (I) is compound(1) and the product compound of formula (II) is compound (2).

Exemplary polypeptides useful in the above process can comprise an aminoacid sequence selected from the group consisting of SEQ ID NO: 4, 6, 8,10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44,46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78. 80.82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112,114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140,142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168,170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196,198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224,226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252,254, 256, 258, 260, 262, 264, and 266, or an engineered CHMO polypeptidethat comprises an amino acid sequence having any one of the sets ofamino acid differences relative to SEQ ID NO: 2 for these polypeptidesthat are listed in Table 2A or 2B.

In some embodiments, the process is used for the conversion of compound(1) to compound (2a) in enantiomeric excess. In some embodiments, theprocess comprises contacting compound (1) with certain monooxygenasepolypeptides described herein in presence of a cofactor under suitablereaction conditions for forming compound (2a) in enantiomeric excess.Exemplary polypeptides useful in the process for preparing compound (2a)in enantiomeric excess can comprise an amino acid sequence selected fromthe group consisting of SEQ ID NO: 4, 6, 160, 162, 164, 166, 168, 170,172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198,200, 202, 204, 206, and 208.

In some embodiments, the process is used for the conversion of compound(1) to compound (2b) in enantiomeric excess. In some embodiments, theprocess comprises contacting compound (1) with certain monooxygenasepolypeptides described herein in presence of a cofactor under suitablereaction conditions for forming compound (2b) in enantiomeric excess.Exemplary polypeptides useful in, the process for preparing compound(2b) in enantiomeric excess can comprise an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 8, 10, 12, 14, 16, 18, 20, 22,24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58,60, 62, 64, 66, 68, 70, 72, 74, 76, 78. 80. 82, 84, 86, 88, 90, 92, 94,96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124,126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152,154, 156, 158, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230,232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258,260, 262, 264, and 266.

In some embodiments, the process can comprise contacting compound (1)with a polypeptide of the disclosure in presence of a cofactor undersuitable reaction conditions to form compound (2b) in at least 90%enantiomeric excess.

Exemplary polypeptides for preparing compound (2b) in at least 90%enantiomeric excess can comprise an amino acid sequence selected fromthe group consisting of SEQ ID NO: 10, 12, 14, 16, 18, 20, 22, 24, 26,28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62,64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98,100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126,128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154,156, 158, 242, 244, 248, 250, 254, 256, 258, 262, and 264.

In some embodiments, the process comprises contacting compound (1) witha polypeptide of the disclosure in presence of a cofactor under suitablereaction conditions to form compound (2b) in at least 99% enantiomericexcess. Exemplary polypeptides for preparing compound (2b) in at least99% enantiomeric excess can comprise an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 16, 18, 20, 22, 24, 26, 28, 30,32, 42, 44, 86, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116,118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144,146, 148, 150, 152, 154, 156, and 158.

In some embodiments, the reaction condition in the process comprises atemperature of 10° C. to 50° C., and in particular 25° C. to 40° C.

In some embodiments, the reaction condition in the process comprises apH of about 8.5 to a pH of about 10, in particular a pH of about 8.5 toabout 9.0.

In some embodiments, the reaction condition in the process comprises apartial pressure of O₂ at greater than atmospheric pressure.Additionally, dissolved molecular oxygen in the process can be increasedby sparging the reaction solution with O₂-containing gas or by use ofbubble-free aeration with O₂-containing gas.

In some embodiments of the process, the reaction condition comprises aco-solvent, such as for example, MeOH, EtOH, isopropanol (IPA), acetone,toluene, MeCN, methyl tert-butyl ether (MTBE), N-methyl-2-pyrrolidone(NMP), dimethylacetamide (DMAc), dimethylformamide (DMF), propyleneglycol, polyethylene glycol (PEG), tetramethylurea,N-ethylpyrollidinone, tetraglyme,1,3-Dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone (DMPU), DMIU,hexamethylphosphoramide (HMPA) and dimethylsulfoxide (DMSO). Theco-solvent can reduce the formation of aggregates which can affect therate and scalability of the process.

In some embodiments, the process further comprises converting NADP⁺ orNAD+ formed from the NADPH or NADH, respectively, with a cofactorregenerating system. The cofactor regenerating system can use anappropriate dehydrogenase, such a glucose dehydrogenase,glucose-phosphate dehydrogenase, formate dehydrogenase, phosphitedehydrogenase, and ketoreductase/alcohol dehydrogenase and correspondingsubstrate, for example, glucose, glucose-6-phosphate, formate,phosphite, or alcohol, respectively. In some embodiments, the co-factorregenerating system is a ketoreductase and a secondary alcohol,particularly isopropanol (IPA).

In a further aspect, the engineered monooxygenase polypeptides can beprovided in the form of kits or arrays, particularly for use in thescreening for activity on other prazole compounds of interest.

5. DETAILED DESCRIPTION

The present disclosure provides efficient biocatalysts capable ofmediating the conversion of the substrate,5-methoxy-2-((4-methoxy-3,5-dimethylpyridin-2-yl)methylthio)-1H-benzo[d]imidazole(“compound (1)”) to the product,5-methoxy-2-((4-methoxy-3,5-dimethylpyridin-2-yl)methylsulfinyl)-1H-benzo[d]imidazole(“compound (2)”). In some embodiments, the biocatalysts are highlystereoselective, capable of mediating the conversion of compound (1) toeither the (S)-isomer of compound (2b) or the (R)-isomer of compound(2a) in enantiomeric excess. The biocatalysts described herein have beendesigned by changing the amino acid sequence of a naturally occurringcyclohexanone monooxygenase (CHMO) to form polypeptides with the desiredenzymatic properties, e.g., enzyme activity, enantioselectivity,byproduct formation, thermostability, solvent stability, and expression.These polypeptides can also be applied to the sulfoxidation of otherprazole compounds structurally similar to esomeprazole.

The detailed description that follow describes these engineeredmonooxygenase polypeptides and processes for carrying out thesulfoxidation of prazole compounds, in particular the conversion ofcompound (1) to compound (2). In some embodiments, the detaileddescription further provides monooxygenase polypeptides for theconversion of compound (1) to compound (2b) in enantiomeric excess.

For the descriptions herein and the appended claims, the singular forms“a”, “an” and “the” include plural referents unless the context clearlyindicates otherwise. Thus, for example, reference to “a polypeptide”includes more than one polypeptide, and reference to “a compound” refersto more than one compound.

Also, the use of “or” means “and/or” unless stated otherwise. Similarly,“comprise,” “comprises,” “comprising” “include,” “includes,” and“including” are interchangeable and not intended to be limiting.

It is to be further understood that where descriptions of variousembodiments use the term “comprising,” those skilled in the art wouldunderstand that in some specific instances, an embodiment can bealternatively described using language “consisting essentially of” or“consisting of:”

It is to be understood that both the foregoing general description,including the drawings, and the following detailed description areexemplary and explanatory only and are not restrictive of thisdisclosure.

5.1 DEFINITIONS

The technical and scientific terms used in the descriptions herein willhave the meanings commonly understood by one of ordinary skill in theart, unless specifically defined otherwise. Accordingly, the followingterms are intended to have the following meanings.

“Protein”, “polypeptide,” and “peptide” are used interchangeably hereinto denote a polymer of at least two amino acids covalently linked by anamide bond, regardless of length or post-translational modification(e.g., glycosylation, phosphorylation, lipidation, myristilation,ubiquitination, etc.). Included within this definition are D- andL-amino acids, and mixtures of D- and L-amino acids.

“Coding sequence” refers to that portion of a nucleic acid (e.g., agene) that encodes an amino acid sequence of a protein.

“Naturally occurring” or “wild-type” refers to the form found in nature.For example, a naturally occurring or wild-type polypeptide orpolynucleotide sequence is a sequence present in an organism that can beisolated from a source in nature and which has not been intentionallymodified by human manipulation.

“Recombinant” or “engineered” or “non-naturally occurring” when usedwith reference to, e.g., a cell, nucleic acid, or polypeptide, refers toa material, or a material corresponding to the natural or native form ofthe material, that has been modified in a manner that would nototherwise exist in nature, or is identical thereto but produced orderived from synthetic materials and/or by manipulation usingrecombinant techniques. Non-limiting examples include, among others,recombinant cells expressing genes that are not found within the native(non-recombinant) form of the cell or express native genes that areotherwise expressed at a different level.

“Percentage of sequence identity,” “percent identity,” and “percentidentical” are used herein to refer to comparisons betweenpolynucleotide sequences or polypeptide sequences, and are determined bycomparing two optimally aligned sequences over a comparison window,wherein the portion of the polynucleotide or polypeptide sequence in thecomparison window may comprise additions or deletions (i.e., gaps) ascompared to the reference sequence for optimal alignment of the twosequences. The percentage is calculated by determining the number ofpositions at which either the identical nucleic acid base or amino acidresidue occurs in both sequences or a nucleic acid base or amino acidresidue is aligned with a gap to yield the number of matched positions,dividing the number of matched positions by the total number ofpositions in the window of comparison and multiplying the result by 100to yield the percentage of sequence identity. Determination of optimalalignment and percent sequence identity is performed using the BLAST andBLAST 2.0 algorithms (see e.g., Altschul et al., 1990, J. Mol. Biol.215: 403-410 and Altschul et al., 1977, Nucleic Acids Res. 3389-3402).Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information website.

Briefly, the BLAST analyses involve first identifying high scoringsequence pairs (HSPs) by identifying short words of length W in thequery sequence, which either match or satisfy some positive-valuedthreshold score T when aligned with a word of the same length in adatabase sequence. T is referred to as, the neighborhood word scorethreshold (Altschul et al, supra). These initial neighborhood word hitsact as seeds for initiating searches to find longer HSPs containingthem. The word hits are then extended in both directions along eachsequence for as far as the cumulative alignment score can be increased.Cumulative scores are calculated using, for nucleotide sequences, theparameters M (reward score for a pair of matching residues; always >0)and N (penalty score for mismatching residues; always <0). For aminoacid sequences, a scoring matrix is used to calculate the cumulativescore. Extension of the word hits in each direction are halted when: thecumulative alignment score falls off by the quantity X from its maximumachieved value; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, T,and X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a wordlength (W) of11, an expectation (E) of 10, M=5, N=−4, and a comparison of bothstrands. For amino acid sequences, the BLASTP program uses as defaults awordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoringmatrix (see Henikoff and Henikoff, 1989, Proc Natl Acad Sci USA89:10915).

Numerous other algorithms are available that function similarly to BLASTin providing percent identity for two sequences. Optimal alignment ofsequences for comparison can be conducted, e.g., by the local homologyalgorithm of Smith and Waterman, 1981, Adv. Appl. Math. 2:482, by thehomology alignment algorithm of Needleman and Wunsch, 1970, J. Mol.Biol. 48:443, by the search for similarity method of Pearson and Lipman,1988, Proc. Natl. Acad. Sci. USA 85:2444, by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe GCG Wisconsin Software Package), or by visual inspection (seegenerally, Current Protocols in Molecular Biology, F. M. Ausubel et al.,eds., Current Protocols, a joint venture between Greene PublishingAssociates, Inc. and John Wiley & Sons, Inc., (1995 Supplement)(Ausubel)). Additionally, determination of sequence alignment andpercent sequence identity can employ the BESTFIT or GAP programs in theGCG Wisconsin Software package (Accelrys, Madison Wis.), using defaultparameters provided.

“Reference sequence” refers to a defined sequence to which anothersequence is compared. A reference sequence may be a subset of a largersequence, for example, a segment of a full-length gene or polypeptidesequence. Generally, a reference sequence is at least 20 nucleotide oramino acid residues in length, at least 25 residues in length, at least50 residues in length, or the full length of the nucleic acid orpolypeptide. Since two polynucleotides or polypeptides may each (1)comprise a sequence (i.e., a portion of the complete sequence) that issimilar between the two sequences, and (2) may further comprise asequence that is divergent between the two sequences, sequencecomparisons between two (or more) polynucleotides or polypeptide aretypically performed by comparing sequences of the two polynucleotidesover a comparison window to identify and compare local regions ofsequence similarity.

The term “reference sequence” is not intended to be limited to wild-typesequences, and can include engineered or altered sequences. For example,in some embodiments, a “reference sequence” can be a previouslyengineered or altered amino acid sequence.

“Comparison window” refers to a conceptual segment of at least about 20contiguous nucleotide positions or amino acids residues wherein asequence may be compared to a reference sequence of at least 20contiguous nucleotides or amino acids and wherein the portion of thesequence in the comparison window may comprise additions or deletions(i.e., gaps) of 20 percent or less as compared to the reference sequence(which does not comprise additions or deletions) for optimal alignmentof the two sequences. The comparison window can be longer than 20contiguous residues, and includes, optionally 30, 40, 50, 100, or longerwindows.

“Corresponding to”, “reference to” or “relative to” when used in thecontext of the numbering of a given amino acid or polynucleotidesequence refers to the numbering of the residues of a specifiedreference sequence when the given amino acid or polynucleotide sequenceis compared to the reference sequence. In other words, the residuenumber or residue position of a given polymer is designated with respectto the reference sequence rather than by the actual numerical positionof the residue within the given amino acid or polynucleotide sequence.For example, a given amino acid sequence, such as that of an engineeredmonooxygenase, can be aligned to a reference sequence by introducinggaps to optimize residue matches between the two sequences. In thesecases, although the gaps are present, the numbering of the residue inthe given amino acid or polynucleotide sequence is made with respect tothe reference sequence to which it has been aligned.

“Stereoselectivity” refers to the preferential formation in a chemicalor enzymatic reaction of one stereoisomer over another.Stereoselectivity can be partial, where the formation of onestereoisomer is favored over the other, or it may be complete where onlyone stereoisomer is formed. When the stereoisomers are enantiomers, thestereoselectivity is referred to as enantioselectivity, the fraction(typically reported as a percentage) of one enantiomer in the sum ofboth. It is commonly alternatively reported in the art (typically as apercentage) as the enantiomeric excess (e.e.) calculated therefromaccording to the formula [major enantiomer−minor enantiomer]/[majorenantiomer+minor enantiomer]. Where the stereoisomers arediastereoisomers, the stereoselectivity is referred to asdiastereoselectivity, the fraction (typically reported as a percentage)of one diastereomer in a mixture of two diastereomers, commonlyalternatively reported as the diastereomeric excess (d.e.). Enantiomericexcess and diastereomeric excess are types of stereomeric excess.

“Highly stereoselective” refers to a chemical or enzymatic reaction thatis capable of converting a substrate (e.g.,5-methoxy-2-((4-methoxy-3,5-dimethylpyridin-2-yl)methylthio)-1H-benzo[d]imidazole)to its corresponding stereoisomeric product (e.g.,(S)-5-methoxy-2-((4-methoxy-3,5-dimethylpyridin-2-yl)methylsulfinyl)-1H-benzo[d]imidazole,or(R)-5-methoxy-2-((4-methoxy-3,5-dimethylpyridin-2-yl)methylsulfinyl)-1H-benzo[d]imidazole)with at least about 85% stereoisomeric excess.

“Increased enzymatic activity” or “increased activity” refers to animproved property of an engineered enzyme, which can be represented byan increase in specific activity (e.g., product produced/time/weightprotein) or an increase in percent conversion of the substrate to theproduct (e.g., percent conversion of starting amount of substrate toproduct in a specified time period using a specified amount ofmonooxygenase) as compared to a reference enzyme. Exemplary methods todetermine enzyme activity are provided in the Examples. Any propertyrelating to enzyme activity may be affected, including the classicalenzyme properties of K_(m), V_(max) or k_(cat), changes of which canlead to increased enzymatic activity. The monooxygenase activity can bemeasured by any one of standard assays used for measuringmonooxygenases, such as change in substrate or product concentration, orchange in concentration of the cofactor (in absence of a cofactorregenerating system). Comparisons of enzyme activities are made using adefined preparation of enzyme, a defined assay under a set condition,and one or more defined substrates, as further described in detailherein. Generally, when enzymes in cell lysates are compared, thenumbers of cells and the amount of protein assayed are determined aswell as use of identical expression systems and identical host cells tominimize variations in amount of enzyme produced by the host cells andpresent in the lysates.

“Conversion” refers to the enzymatic transformation of a substrate tothe corresponding product. “Percent conversion” refers to the percent ofthe substrate that is converted to the product within a period of timeunder specified conditions. Thus, for example, the “enzymatic activity”or “activity” of a monooxygenase polypeptide can be expressed as“percent conversion” of the substrate to the product.

“Thermostable” or “thermal stable” are used interchangeably to refer toa polypeptide that is resistant to inactivation when exposed to a set oftemperature conditions (e.g., 40-80° C.) for a period of time (e.g.,0.5-24 hrs) compared to the untreated enzyme, thus retaining a certainlevel of residual activity (e.g., more than 60% to 80% for example)after exposure to elevated temperatures.

“Solvent stable” refers to a polypeptide that maintains similar activity(more than e.g., 60% to 80%) after exposure to varying concentrations(e.g., 5-99%) of solvent, (e.g., isopropyl alcohol, dimethylsulfoxide,tetrahydrofuran, 2-methyltetrahydrofuran, acetone, toluene,butylacetate, methyl tert-butylether, acetonitrile, etc.) for a periodof time (e.g., 0.5-24 hrs) compared to the untreated enzyme.

“Hydrophilic Amino Acid or Residue” refers to an amino acid or residuehaving a side chain exhibiting a hydrophobicity of less than zeroaccording to the normalized consensus hydrophobicity scale of Eisenberget al., 1984, J. Mol. Biol. 179:125-142. Genetically encoded hydrophilicamino acids include Thr (F), Ser (S), His (H), Glu (E), Asn (N), Gln(O), Asp (D), Lys (K) and Arg (R).

“Acidic Amino Acid or Residue” refers to a hydrophilic amino acid orresidue having a side chain exhibiting a pK value of less than about 6when the amino acid is included in a peptide or polypeptide. Acidicamino acids typically have negatively charged side chains atphysiological pH due to loss of a hydrogen ion. Genetically encodedacidic amino acids include Glu (E) and Asp (D).

“Basic Amino Acid or Residue” refers to a hydrophilic amino acid orresidue having a side chain exhibiting a pK value of greater than about6 when the amino acid is included in a peptide or polypeptide. Basicamino acids typically have positively charged side chains atphysiological pH due to association with hydronium ion. Geneticallyencoded basic amino acids include Arg (R) and Lys (K).

“Polar Amino Acid or Residue” refers to a hydrophilic amino acid orresidue having a side chain that is uncharged at physiological pH, butwhich has at least one bond in which the pair of electrons shared incommon by two atoms is held more closely by one of the atoms.Genetically encoded polar amino acids include Asn (N), Gln (O), Ser (S)and Thr (T).

“Hydrophobic Amino Acid or Residue” refers to an amino acid or residuehaving a side chain exhibiting a hydrophobicity of greater than zeroaccording to the normalized consensus hydrophobicity scale of Eisenberget al., 1984, J. Mol. Biol. 179:125-142. Genetically encoded hydrophobicamino acids include Pro (P), Ile (1), Phe (F), Val (V), Leu (L), Trp(W), Met (M), Ala (A) and Tyr (Y).

“Aromatic Amino Acid or Residue” refers to a hydrophilic or hydrophobicamino acid or residue having a side chain that includes at least onearomatic or heteroaromatic ring. Genetically encoded aromatic aminoacids include Phe (F), Tyr (Y) and Trp (W). Although owing to the itsheteroaromatic ring side chain His (H) is classified as an aromaticresidue, it may also be classified as a basic residue owing to pKa ofits heteroaromatic nitrogen atom.

“Non-polar Amino Acid or Residue” refers to a hydrophobic amino acid orresidue having a side chain that is uncharged at physiological pH andwhich has bonds in which the pair of electrons shared in common by twoatoms is generally held equally by each of the two atoms (i.e., the sidechain is not polar). Genetically encoded non-polar amino acids includeGly (G), Leu (L), Val (V), Ile (I), Met (M) and Ala (A).

“Aliphatic Amino Acid or Residue” refers to a hydrophobic amino acid orresidue having an aliphatic hydrocarbon side chain. Genetically encodedaliphatic amino acids include Ala (A), Val (V), Leu (L) and Ile (I).

The amino acid Cys (C) is unique in that it can form disulfide bridgeswith other Cys (C) amino acids or other sulfanyl- orsulfhydryl-containing amino acids. The ability of Cys (and other aminoacids with —SH containing side chains) to exist in a polypeptide ineither the reduced free —SH or oxidized disulfide-bridged form affectswhether it contributes net hydrophobic or hydrophilic character to thepolypeptide. While Cys exhibits a hydrophobicity of 0.29 according tothe normalized consensus scale of Eisenberg (Eisenberg et al., 1984,supra), it is to be understood that for purposes of the presentdisclosure, Cys is classified into its own unique group.

The amino acid Pro (P) is conformationally constrained nature. Althoughit has hydrophobic properties, as used herein, Pro (P) or other similarresidues is classified as a “conformationally constrained”.

“Hydroxyl-containing Amino Acid or Residue” refers to an amino acid orresidue containing a hydroxyl (—OH) moiety. Genetically-encodedhydroxyl-containing amino acids include Ser (S) and Thr (T). While L-Tyr(Y) contains a hydroxyl moiety, it is classified herein as an aromaticamino acid or residue.

“Amino acid difference” or “residue difference” refers to a change inthe residue at a specified position of a polypeptide sequence whencompared to a reference sequence. For example, a residue difference atposition X3, where the reference sequence has a glutamine, refers to achange of the residue at position X3 to any residue other thanglutamine. As disclosed herein, an enzyme can include one or moreresidue differences relative to a reference sequence, where multipleresidue differences typically are indicated by a list of the specifiedpositions where changes are made relative to the reference sequence.

“Conservative amino acid substitutions” refer to the interchangeabilityof residues having similar side chains, and thus typically involvessubstitution of the amino acid in the polypeptide with amino acidswithin the same or similar defined class of amino acids. By way ofexample and not limitation, an amino acid with an aliphatic side chainmay be substituted with another aliphatic amino acid, e.g., alanine,valine, leucine, and isoleucine; an amino acid with hydroxyl side chainis substituted with another amino acid with a hydroxyl side chain, e.g.,serine and threonine; an amino acids having aromatic side chains issubstituted with another amino acid having an aromatic side chain, e.g.,phenylalanine, tyrosine, tryptophan, and histidine; an amino acid with abasic side chain is substituted with another amino acid with a basisside chain, e.g., lysine and arginine; an amino acid with an acidic sidechain is substituted with another amino acid with an acidic side chain,e.g., aspartic acid or glutamic acid; and a hydrophobic or hydrophilicamino acid is replaced with another hydrophobic or hydrophilic aminoacid, respectively. Exemplary conservative substitutions are providedbelow:

Residue Possible Conservative Substitutions A, L, V, I Other aliphatic(A, L, V, I) Other non-polar (A, L, V, I, G, M) G, M Other non-polar (A,L, V, I, G, M) D, E Other acidic (D, E) K, R Other basic (K, R) N, Q, S,T Other polar H, Y, W, F Other aromatic (H, Y, W, F) C None P None

“Non-conservative substitution” refers to substitution of an amino acidin the polypeptide with an amino acid with significantly differing sidechain properties. Non-conservative substitutions may use amino acidsbetween, rather than within, the defined groups and affects (a) thestructure of the peptide backbone in the area of the substitution (e.g.,proline for glycine) (b) the charge or hydrophobicity, or (c) the bulkof the side chain. By way of example and not limitation, an exemplarynon-conservative substitution can be an acidic amino acid substitutedwith a basic or aliphatic amino acid; an aromatic amino acid substitutedwith a small amino acid; and a hydrophilic amino acid substituted with ahydrophobic amino acid.

“Deletion” refers to modification of the polypeptide by removal of oneor more amino acids from the reference polypeptide. Deletions cancomprise removal of 1 or more amino acids, 2 or more amino acids, 5 ormore amino acids, 10 or more amino acids, 15 or more amino acids, or 20or more amino acids, up to 10% of the total number of amino acids, or upto 20% of the total number of amino acids making up the polypeptidewhile retaining enzymatic activity and/or retaining the improvedproperties of an engineered monooxygenase enzyme. Deletions can bedirected to the internal portions and/or terminal portions of thepolypeptide. In various embodiments, the deletion can comprise acontinuous segment or can be discontinuous.

“Insertion” refers to modification of the polypeptide by addition of oneor more amino acids to the reference polypeptide. In some embodiments,the improved engineered monooxygenase enzymes comprise insertions of oneor more amino acids to the naturally occurring cyclohexanonemonooxygenase polypeptide as well as insertions of one or more aminoacids to other improved monooxygenase polypeptides. Insertions can be inthe internal portions of the polypeptide, or to the carboxy or aminoterminus. Insertions as used herein include fusion proteins as is knownin the art. The insertion can be a contiguous segment of amino acids orseparated by one or more of the amino acids in the naturally occurringpolypeptide.

“Fragment” as used herein refers to a polypeptide that has anamino-terminal and/or carboxy-terminal deletion, but where the remainingamino acid sequence is identical to the corresponding positions in thesequence. Fragments can typically have about 80%, 90%, 95%, 98%, and 99%of the full-length monooxygenase polypeptide, for example thepolypeptide of SEQ ID NO:2.

“Isolated polypeptide” refers to a polypeptide which is substantiallyseparated from other contaminants that naturally accompany it, e.g.,protein, lipids, and polynucleotides. The term embraces polypeptideswhich have been removed or purified from their naturally-occurringenvironment or expression system (e.g., host cell or in vitrosynthesis). The improved monooxygenase enzymes may be present within acell, present in the cellular medium, or prepared in various forms, suchas lysates or isolated preparations. As such, in some embodiments, theimproved monooxygenase enzyme can be an isolated polypeptide.

“Substantially pure polypeptide” refers to a composition in which thepolypeptide species is the predominant species present (i.e., on a molaror weight basis it is more abundant than any other individualmacromolecular species in the composition), and is generally asubstantially purified composition when the object species comprises atleast about 50 percent of the macromolecular species present by mole or% weight. Generally, a substantially pure monooxygenase composition willcomprise about 60% or more, about 70% or more, about 80% or more, about90% or more, about 95% or more, and about 98% or more of allmacromolecular species by mole or % weight present in the composition.Solvent species, small molecules (<500 Daltons), and elemental ionspecies are not considered macromolecular species. In some embodiments,the isolated improved monooxygenase polypeptide is a substantially purepolypeptide composition.

“Heterologous” polynucleotide refers to any polynucleotide that isintroduced into a host cell by laboratory techniques, and includespolynucleotides that are removed from a host cell, subjected tolaboratory manipulation, and then reintroduced into a host cell.

“Codon optimized” refers to changes in the codons of the polynucleotideencoding a protein to those preferentially used in a particular organismsuch that the encoded protein is efficiently expressed in the organismof interest. In some embodiments, the polynucleotides encoding themonooxygenase enzymes may be codon optimized for optimal production fromthe host organism selected for expression.

“Control sequence” is defined herein to include all components, whichare necessary or advantageous for the expression of a polynucleotideand/or polypeptide of the present disclosure. Each control sequence maybe native or foreign to the polynucleotide of interest. Such controlsequences include, but are not limited to, a leader, polyadenylationsequence, propeptide sequence, promoter, signal peptide sequence, andtranscription terminator.

“Operably linked” is defined herein as a configuration in which acontrol sequence is appropriately placed (i.e., in a functionalrelationship) at a position relative to a polynucleotide of interestsuch that the control sequence directs or regulates the expression ofthe polynucleotide and/or polypeptide of interest.

“Alkyl” by itself or as part of another substituent refers to asaturated or unsaturated branched, straight-chain or cyclic monovalenthydrocarbon radical having the stated number of carbon atoms (i.e.,C₁-C₆ means one to six carbon atoms) that is derived by the removal ofone hydrogen atom from a single carbon atom of a parent alkane, alkeneor alkyne. The term “alkyl” is specifically intended to include groupshaving any degree or level of saturation, i.e., groups havingexclusively single carbon-carbon bonds, groups having one or more doublecarbon-carbon bonds, groups having one or more triple carbon-carbonbonds and groups having mixtures of single, double and triplecarbon-carbon bonds. Where a specific level of saturation is intended,the expressions “alkanyl,” “alkenyl,” and “alkynyl” are used. Theexpression “lower alkyl” refers to alkyl groups composed of from 1 to 6carbon atoms (C₁-C₆), preferably 1-4 carbon atoms (C₁-C₄).

“Alkanyl” by itself or as part of another substituent refers to asaturated branched, straight-chain or cyclic alkyl derived by theremoval of one hydrogen atom from a single carbon atom of a parentalkane. Alkanyl groups include, but are not limited to, methanyl;ethanyl; propanyls such as propan-1-yl, propan-2-yl (isopropyl),cyclopropan-1-yl, etc.; butanyls such as butan-1-yl, butan-2-yl(sec-butyl), 2-methyl-propan-1-yl(isobutyl), 2-methyl-propan-2-yl(1-butyl), cyclobutan-1-yl, etc.; and the like. In some embodiments, thealkanyl groups are (C₁-C₆) alkyl.

“Alkenyl” by itself or as part of another substituent refers to anunsaturated branched, straight-chain or cyclic alkyl having at least onecarbon-carbon double bond derived by the removal of one hydrogen atomfrom a single carbon atom of a parent alkene. The group may be in eitherthe cis or trans conformation about the double bond(s). In someembodiments, the alkenyl group is (C₂-C₆) alkenyl.

“Alkynyl” by itself or as part of another substituent refers to anunsaturated branched, straight-chain or cyclic alkyl having at least onecarbon-carbon triple bond derived by the removal of one hydrogen atomfrom a single carbon atom of a parent alkyne. In some embodiments, thealkynyl group is (C₂-C₆) alkynyl.

“Alkoxy” by itself or as part of another substituent refers to —OR^(a),where R^(a) represents an alkyl or cycloalkyl group as defined herein.Typical alkoxy groups include, but are not limited to, methoxy, ethoxy,propoxy, butoxy, cyclohexyloxy, and the like.

“Amino” by itself or as part of another substituent refers to the group—NH₂. Substituted amino refers to the group —NHR^(b), NR^(b)R^(b), andNR^(b)R^(b)R^(b) where each R^(b) is independently selected fromsubstituted or unsubstituted alkyl, cycloalkyl, cycloheteroalkyl,alkoxy, aryl, heteroaryl, heteroarylalkyl, acyl, alkoxycarbonyl,sulfanyl, sulfinyl, sulfonyl, and the like. Typical amino groupsinclude, but are limited to, dimethylamino, diethylamino,trimethylamino, triethylamino, methylysulfonylamino,furanyl-oxy-sulfamino, and the like.

“Heteroalkyl,” Heteroalkanyl,” Heteroalkenyl,” and Heteroalkynyl,” bythemselves or as part of another substituent refer to alkyl, alkanyl,alkenyl, and alkynyl groups, respectively, in which one or more of thecarbon atoms are each independently replaced with the same or differentheteroatoms or heteroatom groups. Heteroatoms and/or heteroatom groupswhich can replace the carbon atoms include, but are not limited to, —O—,—S—, —S—O—, —NR^(c), —PH—, —S(O)—, —S(O)₂—, —S(O)NRS—, —S(O)₂NR^(c)—,and the like, including combinations thereof, where each R^(c)C isindependently hydrogen or (C₁-C₆) alkyl.

“Cycloalkyl” and “Heterocycloalkyl” by themselves or as part of anothersubstituent refer to cyclic versions of “alkyl” and “heteroalkyl”groups, respectively. For heteroalkyl groups, a heteroatom can occupythe position that is attached to the remainder of the molecule. Typicalcycloalkyl groups include, but are not limited to, cyclopropyl;cyclobutyls such as cyclobutanyl and cyclobutenyl; cyclopentyls such ascyclopentanyl and cyclopentenyl; cyclohexyls such as cyclohexanyl andcyclohexenyl; and the like. Typical heterocycloalkyl groups include, butare not limited to, tetrahydrofuranyl (e.g., tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, etc.), piperidinyl (e.g., piperidin-1-yl,piperidin-2-yl, etc.), morpholinyl (e.g., morpholin-3-yl,morpholin-4-yl, etc.), piperazinyl (e.g., piperazin-1-yl,piperazin-2-yl, etc.), and the like.

“Aryl” by itself or as part of another substituent refers to amonovalent aromatic hydrocarbon group having the stated number of carbonatoms (i.e., C₅-C₁₅ means from 5 to 15 carbon atoms) derived by theremoval of one hydrogen atom from a single carbon atom of a parentaromatic ring system. In some embodiments, the aryl group is (C₅-C₁₀)aryl, with (C₅-C₈) being even more preferred. In some embodiments, thearyls are cyclopentadienyl, phenyl and naphthyl.

“Heteroaryl” by itself or as part of another substituent refers to amonovalent heteroaromatic group having the stated number of ring atoms(e.g., “5-14 membered” means from 5 to 14 ring atoms) derived by theremoval of one hydrogen atom from a single atom of a parentheteroaromatic ring system. In some embodiments, the heteroaryl group isa 5-10 membered heteroaryl. In some embodiments, the heteroaryl group isa 5-8 membered heteroaryl.

“Halogen” or “halo” by themselves or as part of another substituent,unless otherwise stated, refer to fluoro, chloro, bromo and iodo.

“Haloalkyl” by itself or as part of another substituent refers to analkyl group in which one or more of the hydrogen atoms is replaced witha halogen. Thus, the term “haloalkyl” is meant to includemonohaloalkyls, dihaloalkyls, trihaloalkyls, etc. up to perhaloalkyls.For example, the expression “(C₁-C₂) haloalkyl” includes 1-fluoromethyl,difluoromethyl, trifluoromethyl, 1-fluoroethyl, 1,1-difluoroethyl,1,2-difluoroethyl, 1,1,1-trifluoroethyl, perfluoroethyl, etc.

“Optionally substituted” as used herein means one or more hydrogen atoms(e.g., 1, 2, 3, 4, 5, or 6 hydrogen atoms) of the group can each bereplaced with a substituent atom or group. Each substituent can be thesame or different. Examples of suitable substituents include, but arenot limited to, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl,cycloheteroalkyl, heteroaryl, OR^(d) (e.g., hydroxyl, alkoxy (e.g.,methoxy, ethoxy, and propoxy), aryloxy, heteroaryloxy, aralkyloxy,ether, ester, carbamate, etc.), hydroxyalkyl, alkoxycarbonyl,alkoxyalkoxy, perhaloalkyl, perfluoroalkyl (e.g., CF₃, CF₂, CF₃),perfluoroalkoxy (e.g., OCF₃, OCF₂CF₃), alkoxyalkyl, SR^(d) (e.g., thiol,alkylthio, arylthio, heteroarylthio, aralkylthio, etc.), S(O)R^(d),SO₂R^(d)NR^(d)R^(e) (e.g., primary amine (i.e., NH₂), secondary amine,tertiary amine, amide, carbamate, urea, etc.), hydrazide, halide,nitrile, nitro, sulfide, sulfoxide, sulfone, sulfonamide, thiol,carboxy, aldehyde, keto, carboxylic acid, ester, amide, imine, andimide, including seleno and thio derivatives thereof, wherein each ofthe substituents can be optionally further substituted. In someembodiments, the number of optional substituents is 1 to 5, wherein thesubstituents are groups as defined herein. Preferably, 1-3 optionalsubstituents can be present. In embodiments in which a functional groupwith an aromatic carbon ring is substituted, such substitutions willtypically number less than about 10 substitutions, more preferably about1 to 5, with about 1 to 3 substitutions being preferred. Generally, asubstituent is selected that does not act as a substrate for themonooxygenases of the present disclosure.

“Substituted alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl”refers to an alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroarylgroup in which one or more hydrogen atoms is replaced with anothersubstituent group.

5.2 DETAILED DESCRIPTION

Cyclohexanone monooxygenases (CHMO) were originally identified for theirability to carry out the conversion of cyclohexanone toepsilon-caprolactone, a seven membered cyclic product, as illustrated inthe following reaction scheme:

The biocatalytic reaction uses O₂ and a co-factor NAPDH to generate thecaprolactone, oxidized cofactor NADP+, and H₂O. Cyclohexanonemonooxygenases are flavin dependent enzymes and contain a flavinprosthetic group, generally flavin adenine dinucleotide (FAD). This FADprosthetic group is bound to the enzyme and, without being bound bytheory, believed to participate in the catalytic reaction by forming aperoxyflavin intermediate (see, e.g., Sheng et al., 2001, Biochemistry40(37):11156-67; Malito et al., 2004, Pro. Natl Acad Sci USA101(36):13157-13162).

Cyclohexanone monooxygenases have also been used as biocatalysts for theenantioselective air-oxidation of prochiral thioethers to form chiralsulfoxides (see, e.g., Light et al., 1982, Biochemistry, 21(10):2490-8).An example of this reaction using a cyclohexanone monooxygenase isdescribed in Reetz et al., 2004, Angew. Chem. Int. Ed. 43:4078-4081:

Cyclohexanone monooxygenases can recognize a variety of aryl-alkylsulfide substrates, examples of which are described in Pasta et al.,1995, Tetrahedron: Asymmetry 6(4):933-936; Yeung and Rettie, 2005,“Prochiral Sulfoxidation as a probe for Flavin-ContainingMonooxygenases, In Methods in Molecular Biology: Cytochrome P950Protocols 320:163-172; Colonna et al., 2000, Chirality 13(1):40-42; andAlphand et al., 2003, Trends Biotechnology 21(7):318-323.

Pyrmetazole, with the chemical name of5-methoxy-2-((4-methoxy-3,5-dimethylpyridin-2-yl)methylthio)-1H-benzo[d]imidazole(also referred to herein as “compound (1)”, is an intermediate in thesynthesis of esomeprazole, with the chemical name of(S)-5-methoxy-2-((4-methoxy-3,5-dimethylpyridin-2-yl)methylsulfinyl)-1H-benzo[d]imidazole(also referred to herein as “compound (2b)”). As noted above,esomeprazole is the S-isomer of omeprazole, which is a racemic mixturecontaining the S and R isomers. Omeprazole and esomeprazole areinhibitors of H⁺K⁺ ATPases, which are proton transporters involved inthe production of acid in the stomach. Omeprazole and esomeprazole areprescribed for the treatment of ulcers, gastroesophageal reflux disease,and Zollinger-Ellison syndrome. The current synthesis of esomeprazoleinvolves a Kagan-Sharpless type chemical oxidation of sulfides tosulfoxides (see Cotton et al., 2000, Tetrahedron: Asymmetry 11: 3819).While biocatalytic oxidation of pyrmetazole mediated by cyclohexanonemonooxygenases is described in U.S. Pat. No. 5,840,552, the process usedwhole cell preparations and resulted in low yield of product. Inparticular, preparations of Acinetobacter NCIMB9871 displayed loweractivity toward pyrmetazole than preparations of other organisms.

In the present disclosure, engineered monooxygenase polypeptides havebeen designed starting from the cyclohexanone monooxygenase ofAcinetobacter NCIMB9871 to efficiently oxidize pyrmetazole (“compound(1)”) to the sulfoxide product (“compound (2)”). The naturally occurringcyclohexanone monooxygenase is inefficient in catalyzing this reaction.In some embodiments, the engineered polypeptides are further capable ofenantioselectively converting pyrmetazole to either the (R) or (5) formof omeprazole in enantiomeric excess. In some embodiments, theseengineered biocatalysts provide a highly efficient process for producingesomeprazole as substantially enantiomerically pure preparations. Theseengineered monooxygenase polypeptides can also be applied to thesulfoxidation of compounds structurally similar to pyrmetazole.

Accordingly, in some embodiments, the present disclosure providespolypeptides capable of converting compound (1) to compound (2), asillustrated in reaction Scheme 1 below, at a rate that is improved overthe monooxygenase of SEQ ID NO:2:

In some embodiments, the conversion rate of the engineered monooxygenaseis greater than 1.5 fold the conversion rate of the monooxygenase of SEQID NO: 2.

Generally, the oxidation occurs in presence of molecular oxygen O₂ andan electron donor, such as cofactor NADPH or NADH. For purposes ofcomparing activities to a reference monooxygenase, such as SEQ ID NO:2,the cofactor NADPH is used. As further discussed below, the reaction canbe carried out in presence of a co-factor regenerating system, forexample an appropriate dehydrogenase (e.g., glucose dehydrogenase,formate dehydrogenase, phosphite dehydrogenase, or ketoreductase/alcoholdehydrogenase) and a suitable dehydrogenase substrate, such as glucose,glucose-6-phosphate, formate, phosphite, or an alcohol, e.g.,isopropanol.

In some embodiments, the polypeptide is capable of converting compound(1) to compound (2) at a rate that is greater than 1.5 fold the rate ofthe monooxygenase polypeptide of SEQ ID NO:2 and comprises an amino acidsequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to a referencesequence selected from the group consisting of SEQ ID NO: 4, 6, 10, 22,52, 76, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112,114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140,142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168,170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196,198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224,226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252,254, 256, 258, 260, 262, 264, and 266. In particular, the referencesequence is SEQ ID NO: 116, 124, 130, 138, or 158.

Some embodiments of the engineered monooxygenase polypeptides arecapable of converting compound (1) to(R)-5-methoxy-2-((4-methoxy-3,5-dimethylpyridin-2-yl)methylsulfinyl)-1H-benzo[d]imidazole(“compound (2a)”) in enantiomeric excess while other embodiments of themonooxygenase polypeptides are capable of converting compound (1) to(S)-5-methoxy-2-((4-methoxy-3,5-dimethylpyridin-2-yl)methylsulfinyl)-1H-benzo[d]imidazole(“compound (2b)”) in enantiomeric excess. As will be apparent to theskilled artisan, the stereoselectivity of the engineered monooxygenasescan be ascertained by using the substrate pyrmetazole and determiningthe amounts of (R) and (S) forms of the products produced. Thestereoselectivity of the monooxygenases towards other prazole substratescan be determined in a similar manner.

In some embodiments, the polypeptides are capable of converting compound(1) to(R)-5-methoxy-2-((4-methoxy-3,5-dimethylpyridin-2-yl)methylsulfinyl)-1H-benzo[d]imidazole(“compound (2a)”) in enantiomeric excess, as illustrated in the reactionScheme 2 below:

In some embodiments, the polypeptide capable of converting compound (1)to compound (2a) in enantiomeric excess comprises an amino acid sequencethat is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or more identical to a reference sequenceselected from the group consisting of SEQ ID NO: 4, 6, 160, 162, 164,166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192,194, 196, 198, 200, 202, 204, 206, and 208. In particular, the referencesequence is SEQ ID NO: 6, 166, 170, 174, 190, 192, 196, 200, 204, or206.

In some embodiments, the polypeptide is capable of converting compound(1) to(S)-5-methoxy-2-((4-methoxy-3,5-dimethylpyridin-2-yl)methylsulfinyl)-1H-benzo[d]imidazole(“compound (2b)”) in enantiomeric excess, as illustrated in the reactionScheme 3 below:

In some embodiments, the polypeptide capable of converting compound (1)to compound (2b) in enantiomeric excess comprises an amino acid sequencethat is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or more identical to a reference sequenceselected from the group consisting of SEQ ID NO: 8, 10, 22, 52, 76, 86,88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116,118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144,146, 148, 150, 152, 154, 156, 158, 210, 212, 214, 216, 218, 220, 222,224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250,252, 254, 256, 258, 260, 262, 264, and 266. In particular, the referencesequence is SEQ ID NO: 116, 124, 130, 138, or 158.

Since the wild type (WT) cyclohexanone monooxygenase of Acinetobacter spNCIMB9871 does not efficiently convert pyrmetazole to the sulfoxide, thepolypeptides presented herein comprise an amino acid sequence that hasone or more residue differences as compared to the reference sequence ofthe naturally occurring cyclohexanone monooxygenase of Acinetobacter spNCIMB9871 represented by SEQ ID NO:2. The residue differences can benon-conservative changes or conservative changes. In some embodiments,the residue differences can be conservative substitutions,non-conservative substitutions, or a combination of non-conservative andconservative substitutions. For the descriptions of the polypeptidesherein, the amino acid residue position in the reference sequence isdetermined in the monooxygenase polypeptide beginning from theinitiating methionine (M) residue (i.e., M represents residue position1), although it will be understood by the skilled artisan that thisinitiating methionine residue may be removed by biological processingmachinery, such as in a host cell or in vitro translation system, togenerate a mature protein lacking the initiating methionine residue. Thepolypeptide sequence position at which a particular amino acid or aminoacid change (“residue difference”) is present is sometimes describedherein as “Xn”, or “position n”, where n refers to the residue positionwith respect to the reference sequence.

Where applicable, a specific substitution mutation, which is areplacement of the specific residue in a reference sequence with adifferent specified residue may be denoted by the conventional notation“X(number)Y”, where X is the single letter identifier of the residue inthe reference sequence, “number” is the residue position in thereference sequence, and Y is the single letter identifier of the residuesubstitution in the engineered sequence.

In some embodiments, the residue differences as compared to SEQ ID NO:2are present at one or more of the following residue positions: X3; X14;X34; X43; X71; X111; X141; X149, X174; X209; X240; X246; X248; X288;X307; X326; X383; X386; X388; X390; X400; X415; X426; X432; X433; X435;X438; X448; X449; X481; X488; X489; X490; X499; X505; X516; X526; X537;and X540. These residue positions are found to be associated withdesirable changes in enzyme activity, enantioselectivity,sulfone-byproduct formation, thermostability, solvent stability, and/orprotein expression. In some embodiments, the monooxygenase has at leasttwo or more, at least three or more, or at least four or more residuedifferences at the residue positions above as compared to the referencesequence of SEQ ID NO:2.

Based on modeling studies of the cyclohexanone monooxygenase ofAcinetobacter sp NCIMB9871 of SEQ ID NO:2, residue positions X14, X34,X43; X111, X141, X386, X388, X426, X432, X433, X435, and X438 are within8A of the FAD prosthetic group on the enzyme; residue positions X149,X209, X277, X326, X426, X432, X435, X438, X488, X489, and X490 arewithin 8A of enzyme-bound NADPH cofactor; and residue positions X277,X326, X426, X432, X433, X435, X438, X489, X490, and X505 are within 8Aof enzyme-bound pyrmetazole. While these residue positions are in closeproximity to bound substrate, FAD prosthetic group, and co-factor, ithas been found that the amino acid residues at these residue positionsas well as the others above can be varied to alter specific enzymeproperties, including, among others, substrate binding, enzyme activity,enantioselectivity, and thermostability.

In some embodiments, specifically excluded from the monooxygenasepolypeptides of the disclosure are polypeptides having an amino acidsequence that contains the following mutation or set of mutations ascompared to SEQ ID NO:2: D41N and F505Y; K78E and F432S; L143F; L220Q,P428S and T433A; F432S; F432I; L426P and A541V; F432Y and K500R; andL143F, E292G, L435Q, and T464A; D384H; K229I and L248P; Y132C, F246I,V361A, and T415A; and F16L and F277S. These monooxygenases are disclosedin Mihovilovic et al., 2006, Organic Lett. 8(6):1221-1224; Reetz et al.,2004, Angew. Chem. Int. Ed. 43:4075-4078; and Reetz et al., 2004, AngewChem. Int. Ed. 43:4078-4081; the contents of which are incorporatedherein by reference.

In some embodiments, specifically excluded from the monooxygenasepolypeptides of the disclosure are polypeptides having the amino acidsequence disclosed in the following UniProt databank accession numbers:(a) gi|81324523|sp|Q9F7E4|Q9F7E4_(—)9GAMM Cyclohexanone monooxygenase;(b) gi|118066|sp|P12015.2|CYMO_ACISP RecName: Full=Cyclohexanone1,2-monooxygenase; (c) gill 23163966|sp|Q11Z78|Q11Z78—POLSJFlavin-containing monooxygenase FMO; (d) tr|A3U3H1|A3U3H1_(—)9RHOBFlavin-containing monooxygenase FMO:FAD dependent oxidoreductaseOS=Oceanicola batsensis HTCC2597 GN=OB2597_(—)18631 PE=4 SV=1; (e)tr|A3T2M3|A3T2M3_(—)9RHOB Flavin-containing monooxygenase FMO:FADdependent oxidoreductase OS=Sulfitobacter sp. NAS-14.1GN=NAS141_(—)04678 PE=4 SV=1; and (f) tr|A1W7Q21A1W7Q2_ACISJCyclohexanone monooxygenase OS=Acidovorax sp. (strain JS42)GN=Ajs_(—)2102 PE=4 SV=1.

In some embodiments, the residue differences as compared to SEQ ID NO:2at the specified residue positions can be selected from the followingfeatures: residue at position corresponding to X3 is ahydroxyl-containing amino acid residue; residue at positioncorresponding to X14 is an aliphatic amino acid residue; residue atposition corresponding to X34 is a basic amino acid residue; residue atposition corresponding to X43 is an aliphatic amino acid residue;residue at position corresponding to X71 is a non-polar amino acidresidue; residue at position corresponding to X111 is ahydroxyl-containing amino acid residue; residue at positioncorresponding to X141 is an aliphatic amino acid residue; residue atposition corresponding to X149 is an aliphatic or aromatic amino acidresidue; residue at position corresponding to X174 is an aliphatic aminoacid residue; residue at position corresponding to X209 is a constrainedamino acid residue; residue at position corresponding to X240 is a basicamino acid residue; residue at position corresponding to X246 is anaromatic amino acid residue; residue at position corresponding to X248is a cysteine (C), or an aliphatic, polar or hydroxyl-containing aminoacid residue; residue at position corresponding to X288 is an aliphaticamino acid residue; residue at position corresponding to X307 is a basicamino acid residue; residue at position corresponding to X326 is acysteine (C) or hydroxyl-containing amino acid residue; residue atposition corresponding to X383 is an aliphatic amino acid residue;residue at position corresponding to X386 is a hydroxyl-containing aminoacid residue; residue at position corresponding to X388 is a basic aminoacid residue; residue at position corresponding to X390 is an aliphaticor basic amino acid residue; residue at position corresponding to X400is an aliphatic amino acid residue; residue at position corresponding toX415 is an aliphatic amino acid residue; residue at positioncorresponding to X426 is an aromatic amino acid residue; residue atposition corresponding to X432 is an aliphatic or hydroxyl-containingamino acid residue; residue at position corresponding to X433 is anon-polar or aliphatic amino acid residue; residue at positioncorresponding to X435 is a hydroxyl-containing amino acid residue;residue at position corresponding to X438 is an aliphatic amino acidresidue; residue at position corresponding to X448 is an aromatic oraliphatic amino acid residue; residue at position corresponding to X449is a non-polar, aliphatic or aromatic amino acid residue; residue atresidue position corresponding to X481 is a basic amino acid residue;residue corresponding to X488 is a basic amino acid residue; residue atposition corresponding to X489 is a cysteine (C); residue at positioncorresponding to X490 is a basic amino acid residue; residue at positioncorresponding to X499 is an aliphatic amino acid residue; residue atposition corresponding to X505 is an aliphatic or aromatic amino acidresidue; residue at position corresponding to X516 is an aliphatic aminoacid residue; residue at position corresponding to X526 is an aliphaticamino acid residue; residue at position corresponding to X537 is ahydroxyl-containing residue; and residue at position corresponding toX540 is a polar or aliphatic amino acid residue. In some embodiments,where the amino acid residue of SEQ ID NO:2 at the corresponding residueposition are encompassed within the category of amino acids describedfor the specified position, a different amino acid within that aminoacid category can be used in light of the guidance provided herein.

In some embodiments, the residue differences as compared to SEQ ID NO:2at the specified residue positions can be selected from the followingfeatures: residue at position corresponding to X3 is T; residue atposition corresponding to X14 is A; residue at position corresponding toX34 is K; residue at position corresponding to X43 is G; residue atposition corresponding to X71 is M; residue at position corresponding toX111 is T; residue at position corresponding to X141 is I or V; residueat position corresponding to X149 is V or W; residue at positioncorresponding to X174 is L or I; residue at position corresponding toX209 is P; residue at position corresponding to X240 is F or K; residueat position corresponding to X246 is E, H, Q, S, R, W, or Y; residue atposition corresponding to X248 is C, I, N, V, or S; residue at positioncorresponding to X277 is L, M, or Q; residue at position correspondingto X278 is G, N, or S; residue at position corresponding to X280 is G,or R; residue at position corresponding to X281 is A, or S; residue atposition corresponding to X282 is S; residue at position correspondingto X248 is C, N, V, or S; residue at position corresponding to X288 isI; residue at position corresponding to X307 is R; residue at positioncorresponding to X326 is C or T; residue at position corresponding toX383 is I or G; residue at position corresponding to X386 is S; residueat position corresponding to X388 is K; residue at positioncorresponding to X390 is R or I; residue at position corresponding toX400 is I; residue at position corresponding to X415 is A; residue atposition corresponding to X426 is F; residue at position correspondingto X432 is A, C, D, H, I, K, L, S, T, or Y; residue at positioncorresponding to X433 is A, F, G, K, L, or W; residue at positioncorresponding to X435 is S or A; residue at position corresponding toX438 is I; residue at position corresponding to X448 is V or W; residueat position corresponding to X449 is M, F, or L; residue at positioncorresponding to X481 is K; residue at position corresponding to X488 isF, K, or L; residue at position corresponding to X489 is C; residue atposition corresponding to X490 is R; residue at position correspondingto X499 is L; residue at position corresponding to X505 is W or L;residue at position corresponding to X516 is V; residue at positioncorresponding to X526 is V; residue at position corresponding to X537 isT; and residue corresponding to X540 is Q or A.

In some embodiments, the polypeptide comprises an amino acid sequencehaving one or more residue differences as compared to SEQ ID NO:2 atresidue positions associated with increased enzyme activity, whichpositions are selected from the following: X14, X34, X43, X71, X111,X141, X149, X174, X209, X240, X246, X248, X277, X278, X280; X281; X282;X288, X307, X326, X341, X368, X386, X388, X390, X400, X415, X426, X432,X433, X435, X438, X448, X449, X481, X488, X489, X490, X499, X505, X516,X526, X537, and X540. In some embodiments, the polypeptide amino acidsequence has at least two or more residue differences, at least three ormore residue differences, or at least four or more residue differencesas compared to SEQ ID NO:2 at the residue positions associated withincreased enzyme activity.

In some embodiments, the polypeptide comprises an amino acid sequencehaving one or more residue differences as compared to SEQ ID NO:2 atresidue positions affecting enantioselectivity, which positions areselected from the following: X246; X248; X326; X386; X432; X433; X435;X438; and X448. In some embodiments, the polypeptide amino acid sequencehas at least two or more residue differences, at least three or moreresidue differences, or at least four or more residue differences ascompared to SEQ ID NO: 2 at the residue positions associated withenantioselectivity.

In some embodiments, the polypeptide comprises an amino acid sequencehaving one or more residue differences as compared to SEQ ID NO:2 atresidue positions associated with decreased amounts of sulfone-byproductformation, where the sulfone-byproduct has the structure of compound (3)(shown above). Residue positions associated with decreased amounts ofsulfone-byproduct of compound (3) are selected from the following: X246,X248, X277, and X438. The sulfone-byproduct of compound (3) is anundesirable product of the enzymatic reaction and is readily identifiedby its elution profile on a chromatographic column of3,5-dimethylphenylcarbamate derivatized amylose immobilized on 5-μmsilica (CHIRALPAK IA®). An exemplary solvent condition for resolution ofthe sulfone-byproduct from pyrmetazole and esomeprazole is 45:55Heptane/EtOH, 1.0 mL/min, 40° C., with detection of eluent at 300 nm. Insome embodiments, the polypeptides are capable of producing amounts ofsulfone-byproduct of compound (3) that is decreased by 20% or more, 40%or more, or 90% or more than the amount produced by the polypeptide ofSEQ ID NO:52 under the same assay conditions.

In some embodiments, the polypeptide capable of forming decreased amountof sulfone-byproduct of compound (3) as compared to the polypeptide ofSEQ ID NO:52 comprises an amino acid sequence which includes one or moreof the following features: residue at position corresponding to X246 isY; residue at position corresponding to X248 is S; and residue atposition corresponding to X438 is I.

In some embodiments, the polypeptide comprises an amino acid sequencehaving one or more residue differences as compared to SEQ ID NO:2 atresidue positions associated with increased thermostability and/orsolvent stability, which positions are selected from the following: X43,X71, X111, X149, X174, X307; X341, X368, X388, X390, X400, X449, X481,and X488. Thermostability can be determined by preincubating thepolypeptide at a set of defined temperatures and times, e.g., 4° C.-50°C. for 18-24 hours, followed by measuring the % residual activity of thepolypeptide using a defined assay. Thermostability can then becharacterized as the temperature at which the variant retains 50%residual activity. In some embodiments, the thermostable polypeptideshave at least 50% residual activity under the exemplary preincubationcondition. Accordingly, in some embodiments the polypeptides of thedisclosure exhibit an increased thermostability of retaining 50%residual activity following 18 hours preincubation at 30° C., 32° C.,34° C., 36° C., 38° C., 40° C., 42° C., 44° C., 46° C., 48° C., 50° C.,52° C., or even higher temperatures. Thermostability can also becharacterized as the activity FIOP when the activity assay is carriedout at an elevated temperature, e.g., FIOP activity at 32° C.Accordingly, in some embodiments the polypeptides of the disclosureexhibit an increased thermostability as determined by an increase inactivity FIOP at an elevated temperature 30° C., 32° C., 34° C., 36° C.,38° C., 40° C., 42° C., 44° C., 46° C., 48° C., 50° C., 52° C., or evenhigher temperatures. Similarly, solvent stability can be determined bypreincubating the polypeptide in a defined solvent at a definedtemperature and time, and measuring the % residual activity of thepolypeptide using a defined assay.

In some embodiments, the polypeptide with increased thermostability ascompared to the polypeptide of SEQ ID NO: 6 comprises an amino acidsequence which includes one or more of the following features: residueat position corresponding to X43 is G; residue at position correspondingto X71 is M; residue at position corresponding to X111 is T; residue atposition corresponding to X149 is V or W; residue at positioncorresponding to X174 is L or I, residue at position corresponding toX307 is R; residue at position corresponding to X341 is E; residue atposition corresponding to X368 is N or V; residue at positioncorresponding to X388 is K; residue at position corresponding to X390 isR; residue at position corresponding to X400 is I; residue at positioncorresponding to X449 is M, F or L; residue at position corresponding toX481 is K; and residue at position corresponding to X488 is K.

In some embodiments, the polypeptide comprises an amino acid sequencehaving residue differences as compared to SEQ ID NO:2 at residuepositions associated with increased protein expression, which positionsinclude X3. In particular, the residue at position corresponding to X3is T.

In addition to the residue positions above that affect enzyme activity,enantioselectivity, sulfone-byproduct formation, thermostability,solvent stability, and/or expression, the polypeptide can haveadditional residue differences as compared to SEQ ID NO:2 at otherresidue positions. Residue differences at these residue other positionsprovide for additional variations in the amino acid sequence withoutadversely affecting the ability of the polypeptide to carry out theconversion of compound (1) to compound (2), in particular the conversionto compound (2b) in enantiomeric excess. In some embodiments, thepolypeptide can comprise an amino acid sequence which includes inaddition to the features above for the specified residue positions, oneor more of the following features: residue at position corresponding toX15 is an aliphatic amino acid residue; residue at positioncorresponding to X22 is an aliphatic amino acid residue; residue atposition corresponding to X32 is a polar amino acid residue; residue atposition corresponding to X39 is an aliphatic amino acid residue;residue at position corresponding to X44 is an acidic amino acidresidue; residue at position corresponding to X59 is an constrainedamino acid residue; residue at position corresponding to X74 is analiphatic amino acid residue; residue at position corresponding to X83is an acidic amino acid residue; residue at position corresponding toX92 is a polar amino acid residue; residue at position corresponding toX107 is a basic amino acid residue; residue at position corresponding toX114 is a basic amino acid residue; residue at position corresponding toX123 is an acidic amino acid residue; residue at position correspondingto X146 is a hydroxyl-containing or aliphatic amino acid residue;residue at position corresponding to X155 is a non-polar amino acidresidue; residue at position corresponding to X161 is an aliphatic or anacidic amino acid residue; residue at position corresponding to X176 isan acidic amino acid residue; residue at position corresponding to X194is a hydroxyl-containing amino acid residue; residue at positioncorresponding to X195 is an aliphatic amino acid residue; residue atposition corresponding to X199 is a constrained amino acid residue;residue at position corresponding to X201 is a polar amino acid residue;residue at position corresponding to X244 is an aliphatic amino acidresidue; residue at position corresponding to X245 is an aliphatic aminoacid residue; residue at position corresponding to X329 is a polar aminoacid residue; residue at position corresponding to X330 ishydroxyl-containing or aliphatic amino acid residue; residue at positioncorresponding to X354 is an aliphatic amino acid residue; residue atposition corresponding to X367 is an acidic amino acid residue; residueat position corresponding to X368 is a polar or aliphatic amino acidresidue; residue at position corresponding to X408 is an aliphatic aminoacid residue; residue at position corresponding to X428 is an aliphaticamino acid residue; residue at position corresponding to X451 is a basicamino acid residue; residue at position corresponding to X454 is analiphatic amino acid residue; residue at position corresponding to X459is a basic amino acid residue; residue at position corresponding to X475is an aliphatic amino acid residue; residue at position corresponding toX507 is an aromatic amino acid residue; and residue at positioncorresponding to X532 is a constrained amino acid residue. As notedabove, in some embodiments, where the amino acid residue of SEQ ID NO:2at the corresponding residue position is encompassed within the categoryof amino acids described for the specified positions of the engineeredpolypeptides, a different amino acid within that amino acid category canbe used in light of the guidance provided herein. Without being bound bytheory, the influence of these residue positions on enzyme function isprovided in Table 3.

In some embodiments, the polypeptide amino acid sequence can have forthe specified residue positions, one or more of the following features:residue at position corresponding to X15 is A; residue at positioncorresponding to X22 is A; residue at position corresponding to X32 isN; residue at position corresponding to X38 is E; residue at positioncorresponding to X39 is G; residue at position corresponding to X44 isE; residue at position corresponding to X59 is P; residue at positioncorresponding to X64R; residue at position corresponding to X74 is G;residue at position corresponding to X83 is E; residue at positioncorresponding to X92 is N; residue at position corresponding to X107 isK; residue at position corresponding to X114 is R; residue at positioncorresponding to is D; residue at position corresponding to X143 is N orV; residue at position corresponding to X144 is A; residue at positioncorresponding to X146 is T or V; residue at position corresponding toX149 is F, M, V, or W; residue at position corresponding to X155 is M;residue at position corresponding to X161 is A, V or D; residue atposition corresponding to X176 is D; residue at position correspondingto X194 is E or S; residue at position corresponding to X195 is G;residue at position corresponding to X199 is P; residue at positioncorresponding to X201 is N; residue at position corresponding to X244 isV; residue at position corresponding to X245 is G; residue at positioncorresponding to X272 is R; residue at position corresponding to X312 isQ; residue at position corresponding to X329 is N; residue at positioncorresponding to X330 is S or G; residue at position corresponding toX354 is A; residue at position corresponding to X367 is E; residue atposition corresponding to X368 is N or V; residue at positioncorresponding to X373 is L; residue at position corresponding to X377 isG; residue at position corresponding to X406 is D; residue at positioncorresponding to X408 is A; residue at position corresponding to X428 isL; residue at position corresponding to X451 is R; residue at positioncorresponding to X454 is I; residue at position corresponding to X459 isK; residue at position corresponding to X464 is D; residue at positioncorresponding to X475 is A; residue at position corresponding to X480N;residue at position corresponding to X499 is G or R; residue at positioncorresponding to X507 is F; residue at position corresponding to X512 isN; residue at position corresponding to X532 is P; and residue atposition corresponding to X541 is G.

As will be understood by the skilled artisan, the monooxygenasepolypeptide can have additional residue differences as compared to SEQID NO:2 at residue positions other than those specified above. In someembodiments, the polypeptides can have additionally 1-2, 1-3, 1-4, 1-5,1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20,1-22, 1-24, 1-26, 1-30, 1-35, 1-40 residue differences at other aminoacid residue positions as compared to the reference sequence. In someembodiments, the number of differences can be 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35, and 40 residuedifferences at other residue positions. The residue difference at theseother positions can include conservative changes or non-conservativechanges. In some embodiments, the residue differences can compriseconservative substitutions and non-conservative substitutions ascompared to the monooxygenase of SEQ ID NO:2. In some embodiments, theother residue differences comprise conservative substitutions.

Various combinations of amino acid residue differences as compared toSEQ ID NO:2 at the residue positions defined above can be used to formthe polypeptides with the desired characteristics. As will be apparentto the skilled artisan, some residue positions can have effects on morethan one enzyme property, and therefore can be used to affect more thanone enzyme property. For example, residues affecting enzyme activity andenantioselectivity can be used in combination to form polypeptides withincreased enzymatic activity and enantioselectivity for the conversionof compound (1) to compound (2b), or increased enzymatic activity andenantioselectivity for the conversion of compound (1) to compound (2a).Additionally, residue positions associated with increasedthermostability, increased solvent stability, decreasedsulfone-byproduct formation, and increased protein expression, can beadded to effect changes in the polypeptide with respect to suchproperties.

In view of the above, in some embodiments, a polypeptide capable ofconverting compound (1) to compound (2a) in enantiomeric excess at arate that is greater than 1.5 fold the rate of the monooxygenase of SEQID NO:2 can comprise an amino acid sequence which includes at least twoor more of the following features: residue at position corresponding toX432 is an aliphatic or hydroxyl-containing amino acid residue; residueat position corresponding to X433 is a non-polar or aliphatic amino acidresidue; residue at position corresponding to X435 is ahydroxyl-containing or aliphatic amino acid residue; and residue atposition corresponding to X490 is a basic amino acid residue. In someembodiments, the R-enantioselective monooxygenases have at least threeor more of the above features, or at least all of the above features.

In some embodiments, the polypeptide capable of converting compound (1)to compound (2a) in enantiomeric excess at a rate that is greater than1.5 fold the rate of the monooxygenase of SEQ ID NO:2 can comprise anamino acid sequence which includes at least two or more of the followingfeatures: residue at position corresponding to X432 is A, C, D, H, K, I,S, T, or Y; residue at position corresponding to X433 is A, F, G, K, orW; residue at position corresponding to X435 is A or S; and residue atposition corresponding to X490 is E, P, or R. In some embodiments, theR-enantioselective monooxygenase has an amino acid sequence that has atleast three or more of the above features, or at least all of the abovefeatures.

In some embodiments of the R-enantioselective polypeptides, residuedifferences at other residue positions associated with desirable changesin other enzyme properties, such as enzyme activity, sulfone-byproductformation, thermostability, solvent stability and/or protein expressioncan be present.

In some embodiments of the R-enantioselective polypeptides, thepolypeptide amino acid sequence can, in addition to the features abovefeatures, further include one or more of the following features: residueat position corresponding to X326 is a hydroxyl-containing residue;residue at position corresponding to X329 is a polar residue; residue atposition corresponding to X383 is an aliphatic amino acid residue; andresidue at position corresponding to X451 is a basic amino acid residue.In some embodiments, amino acid residues at the residue positions areselected from the following: residue at position corresponding to X277is V; residue at position corresponding to X278 is H; residue atposition corresponding to X279 is Y; residue at position correspondingto X280 is W; residue at position corresponding to X281 is H; residue atposition corresponding to X326 is A, D, L, S, or T; residue at positioncorresponding to X329 is N; residue at position corresponding to X383 isI; residue at position corresponding to X426 is H, or Q; residue atposition corresponding to X451 is R; and residue at positioncorresponding to X489 is P.

In some embodiments, the polypeptide capable of converting compound (1)to compound (2a) in enantiomeric excess comprises an amino acid sequencewhich includes at least the following features: residue at positioncorresponding to X432 is A; residue at position corresponding to X433 isA; residue at position corresponding to X435 is S; and residue atposition corresponding to X490 is R. In such embodiments, thepolypeptide capable of converting compound (1) to compound (2a) canfurther comprise one or more of the following features: residue atposition corresponding to X277 is V; residue at position correspondingto X278 is H; residue at position corresponding to X279 is Y; residue atposition corresponding to X280 is W; residue at position correspondingto X281 is H; residue at position corresponding to X326 is A, D, L, S,or T; residue at position corresponding to X329 is N; residue atposition corresponding to X383 is I; residue at position correspondingto X426 is H, or Q; residue at position corresponding to X451 is R; andresidue at position corresponding to X489 is P.

In some embodiments, the polypeptide capable of converting compound (1)to compound (2a) in enantiomeric excess comprises an amino acid sequencewhich includes at least the following features: residue at positioncorresponding to X326 is T; residue at position corresponding to X329 isN; residue at position corresponding to X383 is I; residue at positioncorresponding to X432 is S; residue at position corresponding to X433 isG; residue at position corresponding to X435 is S; residue at positioncorresponding to X451 is R; and residue at position corresponding toX490 is R. In such embodiments, the polypeptide capable of convertingcompound (1) to compound (2a) can further comprise one or more of thefollowing features: residue at position corresponding to X277 is V;residue at position corresponding to X278 is H; residue at positioncorresponding to X279 is Y; residue at position corresponding to X280 isW; residue at position corresponding to X281 is H; residue at positioncorresponding to X326 is A, D, L, S, or T; residue at positioncorresponding to X329 is N; residue at position corresponding to X383 isI; residue at position corresponding to X426 is H, or Q; residue atposition corresponding to X451 is R; and residue at positioncorresponding to X489 is P.

In addition to the residue positions above, the R-enantioselectivepolypeptide can have additional residue differences as compared to SEQID NO:2 at other residue positions. In some embodiments, theR-enantioselective polypeptides can have additionally 1-2, 1-3, 1-4,1-5, 1-6, 1-7, 1-8,1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20,1-22, 1-24, 1-26, 1-30, 1-35, 1-40 residue differences at other aminoacid residue positions as compared to the reference sequence. In someembodiments, the number of differences can be 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35, and 40 residuedifferences at other residue positions. The residue difference at theseother positions can include conservative changes or non-conservativechanges. In some embodiments, the residue differences can compriseconservative substitutions and non-conservative substitutions ascompared to the monooxygenase of SEQ ID NO:2. In some embodiments, theseother residue differences comprise conservative substitutions.

As noted above, some embodiments of the monooxygenases disclosed hereinare capable of converting compound (1) to compound (2b) in enantiomericexcess. In some embodiments, these compound (2b) enantioselectivemonooxygenases comprise an amino acid sequence having residuedifferences at residue positions associated with S-enantioselectivity,particularly at residue positions corresponding to X326 and X386.

In some embodiments, the polypeptide capable of converting compound (1)to compound (2b) in enantiomeric excess comprises an amino acid sequencein which at least residue at position corresponding to X326 is acysteine (C).

In some embodiments, the polypeptide capable of converting compound (1)to compound (2b) in enantiomeric excess comprises an amino acid sequencein which at least residue at position corresponding to X386 is ahydroxyl-containing amino acid residue, particularly a S.

In some embodiments, the polypeptide capable of converting compound (1)to compound (2b) in enantiomeric excess comprises an amino acid sequencein which in which at least residue at position corresponding to X326 isa cysteine (C) and residue at position corresponding to X386 is ahydroxyl-containing amino acid residue, particularly a S.

In some embodiments, the S-enantioselective monooxygenase polypeptidecomprises an amino acid sequence having, in addition to residuedifferences associated with S-enantioselectivity above, at least one ormore residue differences at residue positions associated with increasesin enzyme activity for the pyrmetazole substrate and/or furtherincreases in S-enantioselectivity, particularly at residue positionsX432, X433, X435, X438, X448, and X490; and more particularly at leastone or more residue differences at residue positions X432, X433, X435,and X490.

In some embodiments, the polypeptide capable of converting compound (1)to compound (2b) in enantiomeric excess comprises an amino acid sequencewhich includes at least the following features: residue at positioncorresponding to X326 is a cysteine (C); residue at positioncorresponding to X386 is a hydroxyl-containing amino acid residue; andresidue at position corresponding to X432 is an aliphatic orhydroxyl-containing amino acid residue.

In some embodiments, the polypeptide capable of converting compound (1)to compound (2b) in enantiomeric excess comprises an amino acid sequencewhich includes at least the following features: residue at positioncorresponding to X326 is C; residue at position corresponding to X386 isS; and residue at position corresponding to X432 is A, or L.

In some embodiments of the S-enantioselective monooxygenases, inaddition to the features above at positions X326, X386, and X432, thepolypeptide amino acid sequence can further include one or more of thefollowing features: residue at position corresponding to X433 is analiphatic amino acid residue; residue at position corresponding to X435is a hydroxyl-containing amino acid residue; residue at positioncorresponding to X438 is an aliphatic amino acid residue; residue atposition corresponding to X448 is an aliphatic or aromatic amino acidresidue; and residue at position corresponding to X490 is a basic aminoacid residue. In some embodiments of the S-enantioselectivemonooxygenase, the amino acid residues at the residue positionsassociated with enzymatic activity can be selected from the following:residue at position corresponding to X433 is A, L, or V; residue atposition corresponding to X435 is S; residue at position correspondingto X438 is I; residue at position corresponding to X448 is V or W; andresidue at position corresponding to X490 is R.

In some embodiments, the polypeptide capable of converting compound (1)to compound (2b) in enantiomeric excess comprises amino acid sequencewhich includes at least the following features: residue at positioncorresponding to X326 is a cysteine (C); residue at positioncorresponding to X386 is a hydroxyl-containing amino acid residue;residue at position corresponding to X432 is an aliphatic orhydroxyl-containing amino acid residue; residue at positioncorresponding to X433 is an aliphatic amino acid residue, residue atposition corresponding to X435 is a hydroxyl-containing amino acidresidue; and residue at position corresponding to X490 is a basic aminoacid residue.

In some embodiments, the polypeptide capable of converting compound (1)to compound (2b) in enantiomeric excess comprises an amino acid sequencewhich includes at least the following features: residue at positioncorresponding to X326 is C; residue at position corresponding to X386 isS; residue at position corresponding to X432 is A, or L; residue atposition corresponding to X433 is A, L, or V; residue at positioncorresponding to X435 is S; and residue at position corresponding toX490 is R.

As noted above, residue differences as compared to SEQ ID NO:2 at otherresidue positions associated with desirable changes in enzyme activity,enantioselectivity, sulfone-byproduct formation, thermostability,solvent stability, and expression can be used in combination with thefeatures at the specific residue positions described above to formpolypeptides with additional improvements in the biocatalytic conversionof compound (1) to compound (2b), or in some embodiments, thebiocatalytic conversion of compound (1) to compound (2a). In someembodiments, the polypeptide amino acid sequence can include, inaddition to the above features, one or more of the following features:residue at position corresponding to X3 is a hydroxyl-containing aminoacid residue; residue at position corresponding to X14 is an aliphaticamino acid residue; residue at position corresponding to X34 is a basicamino acid residue; residue at position corresponding to X43 is analiphatic amino acid residue; residue at position corresponding to X71is a non-polar amino acid residue; residue at position corresponding toX83 is a an acidic amino acid residue; residue at position correspondingto X111 is a hydroxyl-containing amino acid residue; residue at positioncorresponding to X141 is an aliphatic amino acid residue; residue atposition corresponding to X149 is an aliphatic or aromatic amino acidresidue; residue at position corresponding to X174 is an aliphatic aminoacid residue; residue at position corresponding to X209 is a constrainedamino acid residue; residue at position corresponding to X240 is anaromatic or a basic amino acid residue; residue at positioncorresponding to X246 is an aromatic amino acid residue; residue atposition corresponding to X248 is a cysteine (C), or an aliphatic,polar, or hydroxyl-containing amino acid residue; residue at positioncorresponding to X288 is an aliphatic amino acid residue; residue atposition corresponding to X307 is a cysteine (C) or a basic amino acidresidue; residue at position corresponding to X341 is an acidic aminoacid residue; residue at position corresponding to X388 is a basic aminoacid residue; residue at position corresponding to X390 is an aliphaticor basic amino acid residue; residue at position corresponding to X400is an aliphatic amino acid residue; residue at position corresponding toX415 is an aliphatic amino acid residue; residue at positioncorresponding to X426 is an aromatic amino acid residue; residue atposition corresponding to X449 is a non-polar or aromatic amino acidresidue; residue at position corresponding to X449 is an acidic residue;residue at position corresponding to X481 is a basic amino acid residue;residue at position corresponding to X488 is a basic amino acid residue;residue at position corresponding to X489 is a cysteine (C); residue atposition corresponding to X499 is an aliphatic amino acid residue;residue at position corresponding to X505 is an aliphatic or aromaticamino acid residue; residue at position corresponding to X516 is analiphatic amino acid residue; residue at position corresponding to X526is an aliphatic amino acid residue; residue at position corresponding toX537 is a hydroxyl-containing residue; and residue at positioncorresponding to X540 is a polar or aliphatic amino acid residue.

In some embodiments, the polypeptide amino acid sequence can include atone or more of the specified residue positions the following features:residue at position corresponding to X3 is T; residue at positioncorresponding to X14 is A; residue at position corresponding to X34 isK; residue at position corresponding to X43 is G; residue at positioncorresponding to X71 is M; residue at position corresponding to X83 isE; residue at position corresponding to X111 is T; residue at positioncorresponding to X141 is I or V; residue at position corresponding toX149 is V or W; residue at position corresponding to X174 is L or I;residue at position corresponding to X209 is P; residue at positioncorresponding to X240 is F or K; residue at position corresponding toX246 is E, H, Q, S, R, W, or Y; residue at position corresponding toX248 is C, I, N, V, or S; residue at position corresponding to X277 isL, M, or Q; residue at position corresponding to X278 is G, N, or S;residue at position corresponding to X280 is G, or R; residue atposition corresponding to X281 is A, or S; residue at positioncorresponding to X282 is S; residue at position corresponding to X288 isI; residue at position corresponding to X307 is C or R; residue atposition corresponding to X341 is E; residue at position correspondingto X373 is L; residue at position corresponding to X377 is G; residue atposition corresponding to X388 is K; residue at position correspondingto X390 is R or 1; residue at position corresponding to X400 is I;residue at position corresponding to X415 is A; residue at positioncorresponding to X426 is F; residue at position corresponding to X449 isM, F, or L; residue at position corresponding to X464 is D; residue atposition corresponding to X481 is K; residue at position correspondingto X488 is F, K, or L; residue at position corresponding to X489 is a C;residue at position corresponding to X499 is L; residue at positioncorresponding to X505 is W or L; residue at position corresponding toX516 is V; residue at position corresponding to X526 is V; residue atposition corresponding to X537 is T; and residue at positioncorresponding to X540 is Q or A.

As noted above, in some embodiments, the polypeptide capable ofconverting compound (1) to compound (2b) in enantiomeric excess, or insome embodiments, capable of converting compound (1) to compound (2a) inenantiomeric excess can comprise an amino acid sequence which includes,in addition to the features above for the specified residue positions,residue differences at one or more of the following positions: X15; X22;X32; X38; X39; X44; X59; X64; X74; X92; X107; X114; X123; X143; X144;X146; X149; X155; X161; X176; X194; X195; X199; X201; X244; X245; X246;X272; X278; X279; X280; X281; X282; X312; X329; X330; X341; X354; X367;X368; X406; X408; X428; X451; X454; X459; X464; X475; X480; X499; X507;X512; X532; and X541.

The amino acid residues that can be used at these additional residuepositions are described above. In some embodiments, where the amino acidresidue of SEQ ID NO:2 at the corresponding residue position areencompassed within the category of amino acids described for thespecified positions of the engineered polypeptides, a different aminoacid within that amino acid category can be used in light of theguidance provided herein.

Thus, in some embodiments, the polypeptide capable of convertingcompound (1) to compound (2) can comprise an amino acid sequence whichincludes at least the following features: residue at positioncorresponding to X34 is K; residue at position corresponding to X209 isP; residue at position corresponding to X240 is K; residue at positioncorresponding to X288 is I; residue at position corresponding to X326 isC; residue at position corresponding to X386 is S; residue at positioncorresponding to X415 is A; residue at position corresponding to X432 isL; residue at position corresponding to X433 is A, residue at positioncorresponding to X435 is S; residue at position corresponding to X438 isI; residue at position corresponding to X448 is V; residue at positioncorresponding to X490 is R; residue at position corresponding to X516 isV; and residue at position corresponding to X537 is T.

In some embodiments, the polypeptide capable of converting compound (1)to compound (2b) in enantiomeric excess comprises an amino acid sequencewhich includes at least the following features: residue at positioncorresponding to X14 is A; residue at position corresponding to X34 isK; residue at position corresponding to X141 is V; residue at positioncorresponding to X209 is P; residue at position corresponding to X240 isK; residue at position corresponding to X288 is I; residue at positioncorresponding to X326 is C; residue at position corresponding to X386 isS; residue at position corresponding to X415 is A; residue at positioncorresponding to X426 is F; residue at position corresponding to X432 isL; residue at position corresponding to X433 is A; residue at positioncorresponding to X435 is S; residue at position corresponding to X438 isI; residue at position corresponding to X448 is V; residue at positioncorresponding to X489 is C; residue at position corresponding to X490 isR; residue at position corresponding to X516 is V; and residue atposition corresponding to X537 is T.

In some embodiments, the polypeptide capable of converting compound (1)to compound (2b) in enantiomeric excess comprises an amino acid sequencewhich includes at least the following features: residue at positioncorresponding to X14 is A; residue at position corresponding to X34 isK; residue at position corresponding to X111 is T; residue at positioncorresponding to X141 is V; residue at position corresponding to X209 isP; residue at position corresponding to X240 is K; residue at positioncorresponding to X246 is Y; residue at position corresponding to X288 isI; residue at position corresponding to X307 is R; residue at positioncorresponding to X326 is C; residue at position corresponding to X386 isS; residue at position corresponding to X388 is K; residue at positioncorresponding to X415 is A; residue at position corresponding to X426 isF; residue at position corresponding to X432 is L; residue at positioncorresponding to X433 is A; residue at position corresponding to X435 isS; residue at position corresponding to X438 is I; residue at positioncorresponding to X448 is V; residue at position corresponding to X481 isK; residue at position corresponding to X489 is C; residue at positioncorresponding to X490 is R; residue at position corresponding to X505 isL; residue at position corresponding to X516 is V; and residue atposition corresponding to X537 is T.

In some embodiments, the polypeptide capable of converting compound (1)to compound (2b) in enantiomeric excess comprises an amino acid sequencewhich includes at least the following features: residue at positioncorresponding to X14 is A; residue at position corresponding to X34 isK; residue at position corresponding to X43 is G; residue at positioncorresponding to X71 is M; residue at position corresponding to X111 isT; residue at position corresponding to X141 is V; residue at positioncorresponding to X149 is W; residue at position corresponding to X209 isP; residue at position corresponding to X240 is K; residue at positioncorresponding to X246 is Y; residue at position corresponding to X248 isV; residue at position corresponding to X277 is M; residue at positioncorresponding to X288 is I; residue at position corresponding to X307 isR; residue at position corresponding to X326 is C; residue at positioncorresponding to X341 is E; residue at position corresponding to X386 isS; residue at position corresponding to X388 is K; residue at positioncorresponding to X390 is I; residue at position corresponding to X415 isA; residue at position corresponding to X426 is F; residue at positioncorresponding to X432 is L; residue at position corresponding to X433 isA; residue at position corresponding to X435 is S; residue at positioncorresponding to X438 is I; residue at position corresponding to X448 isV; residue at position corresponding to X464 is D; residue at positioncorresponding to X481 is K; residue at position corresponding to X489 isC; residue at position corresponding to X490 is R; residue at positioncorresponding to X499 is L; residue at position corresponding to X505 isL; residue at position corresponding to X516 is V; residue at positioncorresponding to X526 is V; residue at position corresponding to X537 isT; and residue at position corresponding to X540 is Q.

In some embodiments, the polypeptide capable of converting compound (1)to compound (2b) in enantiomeric excess comprises an amino acid sequencewhich includes at least the following features: residue at positioncorresponding to X14 is A; residue at position corresponding to X34 isK; residue at position corresponding to X43 is G; residue at positioncorresponding to X71 is M; residue at position corresponding to X83 isE; residue at position corresponding to X111 is T; residue at positioncorresponding to X141 is V; residue at position corresponding to X149 isW; residue at position corresponding to X174 is I; residue at positioncorresponding to X209 is P; residue at position corresponding to X240 isF or K; residue at position corresponding to X246 is Y; residue atposition corresponding to X248 is I or V; residue at positioncorresponding to X277 is M; residue at position corresponding to X288 isI; residue at position corresponding to X307 is C or R; residue atposition corresponding to X326 is C; residue at position correspondingto X341 is E; residue at position corresponding to X373 is L; residue atposition corresponding to X377 is G; residue at position correspondingto X383 is G; residue at position corresponding to X386 is S; residue atposition corresponding to X388 is K; residue at position correspondingto X390 is 1; residue at position corresponding to X400 is I; residue atposition corresponding to X415 is A; residue at position correspondingto X426 is F; residue at position corresponding to X432 is L; residue atposition corresponding to X433 is A; residue at position correspondingto X435 is S; residue at position corresponding to X438 is I; residue atposition corresponding to X448 is V; residue at position correspondingto X449 is F; residue at position corresponding to X464 is D; residue atposition corresponding to X481 is K; residue at position correspondingto X488 is K; residue at position corresponding to X489 is C; residue atposition corresponding to X490 is R; residue at position correspondingto X499 is L; residue at position corresponding to X505 is L; residue atposition corresponding to X516 is V; residue at position correspondingto X526 is V; residue at position corresponding to X537 is T; andresidue at position corresponding to X540 is Q.

In some embodiments, the above polypeptides can have additionally 1-2,1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16,1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35, 1-40 residue differences atother amino acid residue positions as compared to the referencesequence. In some embodiments, the number of differences can be 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35,and 40 residue differences at other residue positions. The residuedifference at these other positions can include conservative changes ornon-conservative changes. In some embodiments, the residue differencescan comprise conservative substitutions and non-conservativesubstitutions as compared to the monooxygenase of SEQ ID NO:2. In someembodiments, the residue differences comprise conservativesubstitutions.

In some embodiments, the polypeptide capable of forming compound (2b) inenantiomeric excess comprises an amino acid sequence selected from thegroup consisting of SEQ ID NO: 8, 10, 12, 14, 16, 18, 20, 22, 24, 26,28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62,64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98,100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126,128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154,156, 158, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232,234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260,262, 264, and 266.

In some embodiments, the polypeptide is capable of converting compound(1) to compound (2b) in at least 90% enantiomeric excess. In someembodiments, the polypeptide is capable of forming compound (2b) in atleast 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or moreenantiomeric excess.

In some embodiments, the polypeptide capable of forming compound (2b) inat least 90% enantiomeric excess comprises an amino acid sequence whichincludes at least one or more, at least two or more, at least three ormore, or at least four or more of the following features: residue atposition corresponding to X246 is Y; residue at position correspondingto X248 is S; residue at position corresponding to X326 is C; residue atposition corresponding to X386 is S; residue at position correspondingto X432 is L; residue at position corresponding to X433 is A; residue atposition corresponding to X435 is S; residue at position correspondingto X438 is I; and residue at position corresponding to X448 is V.

In some embodiments, the polypeptide capable of forming compound (2b) inat least 90% enantiomeric excess comprises an amino acid sequence whichincludes at least the following features: residue at positioncorresponding to X326 is C; residue at position corresponding to X386 isS; residue at position corresponding to X438 is I; and residue atposition corresponding to X448 is V.

In some embodiments, the polypeptide capable of forming compound (2b) inat least 90% enantiomeric excess comprises an amino acid sequenceselected from the group consisting of SEQ ID NO: 10, 12, 14, 16, 18, 20,22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56,58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92,94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122,124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150,152, 154, 156, 158, 242, 244, 248, 250, 254, 256, 258, 262, and 264.

In some embodiments, the polypeptide is capable of forming compound (2b)in at least 99% enantiomeric excess and with at least 4-fold theenzymatic activity of the polypeptide of SEQ ID NO:10.

In some embodiments, the polypeptide capable of forming compound (2b) inat least 99% enantiomeric excess and with at least 4 fold or more theenzymatic activity of the polypeptide of SEQ ID NO:10 comprises an aminoacid sequence selected from the group consisting of SEQ ID NO: 16, 18,20, 22, 24, 26, 28, 30, 32, 42, 44, 86, 94, 96, 98, 100, 102, 104, 106,108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134,136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, and 158.

As provided herein, in some embodiments, the monooxygenase polypeptidesare capable of forming decreased amount of sulfone-byproduct of compound(3) as compared to the polypeptide of SEQ ID NO: 52. Exemplarymonooxygenase polypeptides capable of forming decreased amount ofsulfone-byproduct as compared to the polypeptide of SEQ ID NO:52 cancomprise an amino acid sequence selected from the group consisting ofSEQ ID NO: 54, 58, 60, 62, 64, 70, 72, 76, 84, 86, 88, 90, 92, 94, 96,98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124,126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152,154, 156, and 158.

In some embodiments, the polypeptide is capable of formingsulfone-byproduct of compound (3) in an amount that is decreased byabout 20% or higher as compared to the amount produced by thepolypeptide of SEQ ID NO: 52 and comprises an amino acid sequenceselected from the group consisting of SEQ ID NO: 54, 58, 60, 62, 64, 70,72, 76, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110,112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138,140, 142, 144, 146, 148, 150, 152, 154, 156, and 158.

In some embodiments, the polypeptide is capable of formingsulfone-byproduct of compound (3) in an amount that is decreased byabout 40% or higher as compared to the amount produced by thepolypeptide of SEQ ID NO: 52 and comprises an amino acid sequenceselected from the group consisting of SEQ ID NO: 54, 72, 76, 84, 86, 88,90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118,120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146,148, 150, 152, 154, 156, and 158.

In some embodiments, the polypeptide is capable of formingsulfone-byproduct of compound (3) in an amount that is decreased byabout 90% or higher as compared to the amount produced by thepolypeptide of SEQ ID NO:52 and comprises an amino acid sequenceselected from the group consisting of SEQ ID NO: 84, 86, 88, 90, 92, 94,96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124,126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152,154, 156, and 158.

As noted above, in some embodiments, the polypeptide amino acid sequencecan have residue differences as compared to SEQ ID NO: 2 at residuepositions associated with increases in protein expression. Accordingly,in some embodiments, the polypeptide amino acid sequences describedherein can further include at the residue position corresponding to X3ahydroxyl-containing residue. In some embodiments, the residue atposition corresponding to X3 is T. This feature is particularly usefulfor enhanced expression in a bacterial host organism, particularly E.coli.

Table 2A below provides exemplary engineered polypeptides capable ofconverting compound (1) to compound (2b). Odd numbered SEQ ID NOs referto the nucleotide sequence encoding the amino acid sequence provided bythe even numbered SEQ ID NOs. The residue differences are based oncomparison to reference sequence of SEQ ID NO:2, the naturally occurringcyclohexanone monooxygenase of Acinetobacter sp NCIMB9871. Activityimprovements are presented as “fold improvement over parent” or “FIOP”,where the parent polypeptide amino acid sequence used as reference isindicated by the SEQ ID NO in a separate column. The polypeptides of SEQID NO: 4 and 6 have the ability to convert compound (1) to compound (2)but the product has enantiomeric excess of the R-isomer. Themonooxygenase of SEQ ID NO: 4 has about 1.7 fold the activity of SEQ IDNO:2.

The Activity FIOP and enantioselectivity (% ee) of the exemplarynon-naturally occurring monooxygenase polypeptides (“engineered CHMOpolypeptides”) of Table 2A in carrying out the biocatalytic conversionof the substrate compound (1) to the product compound (2) weredetermined using the following general HTP assay conditions: 6 g/Lsubstrate compound (1), 10 μL of lysate of the engineered CHMOpolypeptide, 1 g/L KRED of SEQ ID NO: 268, 0.1 g/L NADP, in a solutionof 50 mM potassium phosphate buffer, 8% (v/v) IPA, 2% acetone, 15%N-methyl-2-pyrrolidone (NMP), pH 9.0, 25° C. reaction temperature and 24h reaction time. Further details of the HTP assay used are described inthe Examples.

TABLE 2A Engineered CHMO polypeptides having improved propertiesActivity FIOP SEQ ID (relative % ee NO Residue Differences to SEQ ID ofS (nt/aa) (relative to SEQ ID NO: 2) NO: 2) Isomer 1/2 NA (wild-type)1.0 ND 3/4 F432A; T433A; L435S; W490R + −95.7 5/6 K326T; L329N; V383I;F432S; T433G; L435A; Q451R; W490R + −98.9 7/8 K326C; N386S; F432A;T433A; L435S; W490R + 6.7  9/10 K326C; N386S; F432A; T433A; L435S;S438I; D448V; W490R + 90.9 11/12 K326C; N386S; F432L; T433A; L435S;S438I; D448V; W490R + 99.1 13/14 K326C; N386S; F432A; T433A; L435S;S438I; D448W; W490R + 92.9 15/16 Q3T; K326C; N386S; F432L; T433A; L435S;S438I; D448V; W490R ++ 99.9 17/18 Q3T; Q34K; W240K; A288I; K326C; N386S;F432L; T433A; L435S; ++ 100.0 S438I; D448V; W490R; A516V 19/20 Q3T;Q34K; E161A; A288I; K326C; N386S; F432L; T433A; L435S; +++ 100.0 S438I;D448V; W490R; I537T 21/22 Q3T; K32N; E161A; F174L; A288I; K326C; N386S;F432L; T433A; +++ 100.0 L435S; S438I; D448V; W490R 23/24 Q3T; Q34K;V195G; W240K; A288I; K326C; N386S; T415A; F432L; +++ 100.0 T433A; L435S;S438I; D448V; W490R; A516V; I537T 25/26 Q3T; Q34K; E123D; W240K; L244V;A288I; K326C; N386S; M390R; +++ 100.0 T415A; F432L; T433A; L435S; S438I;D448V; W490R; A516V 27/28 Q3T; Q34K; W240K; A288I; K326C; N386S; T415A;F432L; T433A; +++ 100.0 L435S; S438I; D448V; W490R; A516V; I537T 29/30Q3T; Q34K; W240K; K326C; N386S; P408A; P428L; F432L; T433V; +++ 101.0L435S; S438I; D448V; W490R; A516V; L532P; P540A 31/32 Q3T; Q34K; A209P;W240K; A288I; K326C; N386S; T415A; F432L; +++ 100.0 T433A; L435S; S438I;D448V; W490R; A516V; I537T 33/34 Q3T; G15A; Q34K; L141I; A199P; H201N;W240K; A288I; K326C; +++ D367E; F368N; N386S; T415A; F432L; T433A;L435S; S438I; D448V; W490R; A516V; I537T 35/36 Q3T; Q34K; L141V; A209P;W240K; A288I; K326C; N386S; T415A; +++ F432L; T433A; L435S; S438I;D448V; W490R; A516V; I537T 37/38 Q3T; Q34K; L141V; A209P; W240K; A288I;K326C; N386S; T415A; +++ F432L; T433A; L435S; S438I; D448V; W490R;A516V; I537T 39/40 Q3T; Q34K; A146T; A209P; W240K; A288I; K326C; N386S;T415A; ++ F432L; T433A; L435S; S438I; D448V; W490R; F505W; A516V; I537T41/42 Q3T; Q34K; N107K; L141V; A209P; W240K; A288I; K326C; N386S; +++T415A; F432L; T433A; L435S; S438I; D448V; W490R; A516V; I537T 43/44 Q3T;V22A; Q34K; G44E; E161V; G176D; A194S; A209P; W240K; +++ 100.0 F277L;A288I; K326C; C330S; V354A; N386S; T415A; F432L; T433A; L435S; S438I;D448V; W490R; A516V; I537T 45/46 Q3T; Q34K; A146T; A209P; W240K; A288I;K326C; V354A; N386S; +++ 97.8 T415A; F432L; T433A; L435S; S438I; D448V;W490R; A516V; I537T 47/48 Q3T; Q34K; A209P; W240K; A288I; K326C; N386S;T415A; F432L; +++ 97.7 T433A; L435S; S438I; D448V; W490R; A516V; I537T49/50 Q3T; Q34K; S74G; Q92N; A146V; L149V; E161D; F174L; A209P; +++ 97.3W240K; A288I; K326C; N386S; T415A; F432L; T433A; L435S; S438I; D448V;W490R; A516V; L532P; I537T 51/52 Q3T; Q34K; L141V; A209P; W240K; A288I;K326C; N386S; T415A; ++++ F432L; T433A; L435S; S438I; D448V; S489C;W490R; A516V; I537T 53/54 Q3T; G14A; Q34K; L141V; A146V; F174L; A209P;W240K; A288I; ++++ K326C; N386S; T415A; F432L; T433A; L435S; S438I;D448V; S489C; W490R; A516V; I537T 55/56 Q3T; G14A; Q34K; L141V; A146V;F174L; A209P; W240K; A245G; ++++ F277L; A288I; K326C; C330G; N386S;T415A; F432L; T433A; L435S; S438I; D448V; W490R; A516V; I537T 57/58 Q3T;G14A; Q34K; L141V; A209P; W240K; A288I; K326C; N386S; ++++ T415A; L426F;F432L; T433A; L435S; S438I; D448V; S489C; W490R; A516V; I537T 59/60 Q3T;G14A; Q34K; L141V; A209P; W240K; A288I; K326C; N386S; ++++ T415A; L426F;F432L; T433A; L435S; S438I; D448V; S489C; W490R; A516V; I537T 61/62 Q3T;G14A; Q34K; L141V; A209P; W240K; A288I; K326C; N386S; ++++ T415A; L426F;F432L; T433A; L435S; S438I; D448V; S489C; W490R; A516V; I537T 63/64 Q3T;G14A; Q34K; L141V; A209P; W240K; A288I; K326C; N386S; ++++ T415A; L426F;F432L; T433A; L435S; S438I; D448V; S489C; W490R; A516V; I537T 65/66 Q3T;Q34K; L141V; F174L; A209P; W240K; A288I; E307R; K326C; ++++ N386S;T415A; F432L; T433A; L435S; S438I; D448V; M481K; S489C; W490R; F505L;A516V; I537T 67/68 Q3T; Q34K; Q83E; L141V; F174L; A209P; W240K; A288I;E307R; K326C; ++++ N386S; T415A; F432L; T433A; L435S; S438I; D448V;M481K; S489C; W490R; F505L; A516V; I537T 69/70 Q3T; G14A; Q34K; L141V;A209P; W240K; A288I; E307R; K326C; ++++ N386S; T415A; L426F; F432L;T433A; L435S; S438I; D448V; M481K; S489C; W490R; F505L; A516V; I537T71/72 Q3T; G14A; Q34K; L141V; A209P; W240K; A288I; E307R; K326C; ++++N386S; T415A; L426F; F432L; T433A; L435S; S438I; D448V; M481K; S489C;W490R; F505L; A516V; I537T 73/74 Q3T; G14A; Q34K; L141V; A209P; W240K;A288I; K326C; N386S; ++++ T415A; L426F; F432L; T433A; L435S; S438I;D448V; C475A; S489C; W490R; A516V; I537T 75/76 Q3T; G14A; Q34K; Q111T;L141V; A209P; W240K; A288I; E307R; +++++ K326C; N386S; V388K; T415A;L426F; F432L; T433A; L435S; S438I; D448V; M481K; S489C; W490R; F505L;A516V; I537T 77/78 Q3T; G14A; Q34K; A43G; Q111T; L141V; A209P; W240K;A288I; +++++ E307R; K326C; N386S; V388K; T415A; L426F; F432L; T433A;L435S; S438I; D448V; M481K; S489C; W490R; F505L; A516V; I537T 79/80 Q3T;G14A; Q34K; A43G; H114R; L141V; I155M; A209P; W240K; +++++ A288I; E307R;K326C; N386S; T415A; L426F; F432L; T433A; L435S; S438I; D448V; T449M;M481K; S489C; W490R; F505L; A516V; I537T 81/82 Q3T; G14A; Q34K; A43G;H114R; L141V; I155M; A209P; W240K; +++++ A288I; E307R; K326C; N386S;T415A; L426F; F432L; T433A; L435S; S438I; D448V; T449M; E459K; M481K;S489C; W490R; F505L; A516V; I537T 83/84 Q3T; G14A; Q34K; A43G; Q111T;L141V; L149W; A209P; W240K; +++++ A288I; E307R; K326C; F368V; N386S;V388K; T415A; L426F; F432L; T433A; L435S; S438I; D448V; M481K; S489C;W490R; F505L; A516V; I537T 85/86 Q3T; G14A; Q34K; Q111T; L141V; A209P;W240K; F246Y; A288I; ++++ 99.4 E307R; K326C; N386S; V388K; T415A; L426F;F432L; T433A; L435S; S438I; D448V; M481K; S489C; W490R; F505L; A516V;I537T 87/88 Q3T; G14A; Q34K; Q111T; L141V; A209P; W240K; F246W; A288I;++++ 98.7 E307R; K326C; N386S; V388K; T415A; L426F; F432L; T433A; L435S;S438I; D448V; M481K; S489C; W490R; F505L; A516V; I537T 89/90 Q3T; G14A;Q34K; Q111T; L141V; A209P; W240K; F277M; A288I; ++++ 98.2 E307R; K326C;N386S; V388K; T415A; L426F; F432L; T433A; L435S; S438I; D448V; M481K;S489C; W490R; F505L; A516V; I537T 91/92 Q3T; G14A; Q34K; Q111T; L141V;A209P; W240K; A288I; E307R; +++++ K326C; N386S; V388K; T415A; L426F;F432L; T433A; L435S; S438I; D448V; T449F; V454I; M481K; S489C; W490R;F505L; A516V; I537T 93/94 Q3T; G14A; Q34K; Q111T; L141V; A209P; W240K;F246Y; L248C; +++++ 99.5 A288I; E307R; K326C; N386S; V388K; T415A;L426F; F432L; T433A; L435S; S438I; D448V; M481K; S489C; W490R; F505L;A516V; I537T 95/96 Q3T; G14A; Q34K; Q111T; L141V; A209P; W240K; F246Y;L248N; +++++ 99.5 A288I; E307R; K326C; N386S; V388K; T415A; L426F;F432L; T433A; L435S; S438I; D448V; M481K; S489C; W490R; F505L; A516V;I537T 97/98 Q3T; G14A; Q34K; Q111T; L141V; A209P; W240K; F246Y; L248V;+++++ 99.3 A288I; E307R; K326C; N386S; V388K; T415A; L426F; F432L;T433A; L435S; S438I; D448V; M481K; S489C; W490R; F505L; A516V; I537T 99/100 Q3T; G14A; Q34K; Q111T; L141V; A209P; W240K; F246Y; L248S; ++++99.4 A288I; E307R; K326C; N386S; V388K; T415A; L426F; F432L; T433A;L435S; S438I; D448V; M481K; S489C; W490R; F505L; A516V; I537T 101/102Q3T; G14A; Q34K; Q111T; L141V; A209P; W240K; F246Y; A288I; ++++ E307R;K326C; N386S; V388K; T415A; L426F; F432L; T433A; L435S; S438I; D448V;M481K; S489C; W490R; F505L; L507F; A516V; I537T 103/104 Q3T; G14A; Q34K;Q111T; L141V; A209P; W240K; F246Y; L248V; +++++ A288I; E307R; K326C;N386S; V388K; T415A; L426F; F432L; T433A; L435S; S438I; D448V; M481K;S489C; W490R; F505L; A516V; E526V; I537T; P540Q 105/106 Q3T; G14A; Q34K;E59P; Q111T; L141V; A209P; W240K; F246Y; ++++ L248V; A288I; E307R;K326C; N386S; V388K; T415A; L426F; F432L; T433A; L435S; S438I; D448V;M481K; S489C; W490R; K499L; F505L; A516V; I537T 107/108 Q3T; G14A; Q34K;Q111T; L141V; A209P; W240K; F246Y; L248V; +++++ F277M; A288I; E307R;K326C; N386S; V388K; T415A; L426F; F432L; T433A; L435S; S438I; D448V;M481K; S489C; W490R; K499L; F505L; A516V; E526V; I537T; P540Q 109/110Q3T; G14A; Q34K; Q111T; L141V; A209P; W240K; F246Y; L248V; +++++ F277M;A288I; E307R; K326C; N386S; V388K; T415A; L426F; F432L; T433A; L435S;S438I; D448V; M481K; S489C; W490R; F505L; A516V; I537T; P540Q 111/112Q3T; G14A; Q34K; Q111T; L141V; A209P; W240K; F246Y; L248V; +++++ F277M;A288I; E307R; K326C; N386S; V388K; T415A; L426F; F432L; T433A; L435S;S438I; D448V; M481K; S489C; W490R; F505L; A516V; I537T; P540Q 113/114Q3T; G14A; Q34K; A43G; Q111T; L141V; L149W; A209P; W240K; +++++ F246Y;L248S; A288I; E307R; K326C; D341E; N386S; V388K; T415A; L426F; F432L;T433A; L435S; S438I; D448V; T449L; M481K; S489C; W490R; F505L; A516V;I537T 115/116 Q3T; G14A; Q34K; A43G; L71M; Q111T; L141V; L149W; A209P;+++++ W240K; F246Y; L248V; F277M; A288I; E307R; K326C; N386S; V388K;M390I; T415A; L426F; F432L; T433A; L435S; S438I; D448V; M481K; S489C;W490R; K499L; F505L; A516V; E526V; I537T; P540Q 117/118 Q3T; G14A; Q34K;A43G; L71M; Q111T; L141V; L149W; F174L; +++++ A209P; W240K; F246Y;L248V; F277M; A288I; E307R; K326C; N386S; V388K; M400I; T415A; L426F;F432L; T433A; L435S; S438I; D448V; M481K; S489C; W490R; K499L; F505L;A516V; E526V; I537T; P540Q 119/120 Q3T; G14A; Q34K; L71M; Q111T; L141V;L149W; F174L; A209P; +++++ W240K; F246Y; L248V; F277M; A288I; E307R;K326C; N386S; V388K; M390I; T415A; L426F; F432L; T433A; L435S; S438I;D448V; M481K; S489C; W490R; K499L; F505L; A516V; E526V; I537T; P540Q121/122 Q3T; G14A; Q34K; A43G; L71M; Q111T; L141V; L149W; F174L; +++++A209P; W240K; F246Y; L248V; F277M; A288I; E307R; K326C; N386S; V388K;M390I; T415A; L426F; F432L; T433A; L435S; S438I; D448V; M481K; S489C;W490R; K499L; F505L; A516V; E526V; I537T; P540Q 123/124 Q3T; G14A; Q34K;A43G; L71M; Q111T; L141V; L149W; F174I; +++++ A209P; W240K; F246Y;L248V; F277M; A288I; E307R; K326C; D341E; V383G; N386S; V388K; M390I;M400I; T415A; L426F; F432L; T433A; L435S; S438I; D448V; T449F; M481K;Q488K; S489C; W490R; K499L; F505L; A516V; E526V; I537T; P540Q 125/126Q3T; G14A; Q34K; A43G; L71M; Q111T; L141V; L149W; A209P; +++++ W240K;F246Y; L248V; F277M; A288I; E307R; K326C; V383G; N386S; V388K; M400I;T415A; L426F; F432L; T433A; L435S; S438I; D448V; T449F; M481K; Q488K;S489C; W490R; K499L; F505L; A516V; E526V; I537T; P540Q 127/128 Q3T;G14A; Q34K; A39G; A43G; L71M; Q111T; L141V; L149W; +++++ A209P; W240K;F246Y; L248V; F277M; A288I; E307R; K326C; V383G; N386S; V388K; M400I;T415A; L426F; F432L; T433A; L435S; S438I; D448V; T449F; M481K; Q488K;S489C; W490R; K499L; F505L; A516V; E526V; I537T; P540Q 129/130 Q3T;G14A; Q34K; A43G; L71M; Q111T; L141V; L149W; A209P; +++++ W240K; F246Y;L248V; F277M; A288I; E307R; K326C; V383G; N386S; V388K; M390I; M400I;T415A; L426F; F432L; T433A; L435S; S438I; D448V; T449L; M481K; Q488K;S489C; W490R; K499L; F505L; A516V; E526V; I537T; P540Q 131/132 Q3T;G14A; Q34K; A43G; L71M; Q111T; L141V; L149W; F174I; +++++ A209P; W240K;F246Y; L248V; F277M; A288I; E307R; K326C; D341E; V383G; N386S; V388K;M390I; M400I; T415A; L426F; F432L; T433A; L435S; S438I; D448V; T449F;M481K; Q488K; S489C; W490R; K499R; F505L; A516V; E526V; I537T; P540Q;133/134 Q3T; G14A; Q34K; A43G; L71M; Q111T; L141V; L149W; F174I; +++++A209P; W240K; A245G; F246Y; L248V; F277M; A288I; E307R; K326C; D341E;V383G; N386S; V388K; M390I; M400I; T415A; L426F; F432L; T433A; L435S;S438I; D448V; T449F; M481K; Q488K; S489C; W490R; K499L; F505L; A516V;E526V; I537T; P540Q; 135/136 Q3T; G14A; Q34K; K38E; A43G; C64R; L71M;Q83E; Q111T; +++++ L141V; L149W; F174I; A209P; W240K; A245G; F246Y;L248V; F277M; A288I; E307C; K326C; D341E; M373L; V383G; N386S; V388K;M390I; M400I; E406D; T415A; L426F; F432L; T433A; L435S; S438I; D448V;T449F; T464D; M481K; Q488K; S489C; W490R; F505L; A516V; E526V; I537T;P540Q; 137/138 Q3T; G14A; Q34K; A43G; L71M; Q83E; Q111T; L141V; L149W;+++++ F174I; A209P; W240K; A245G; F246Y; L248V; F277M; A288I; E307C;K326C; D341E; M373L; V383G; N386S; V388K; M390I; M400I; T415A; L426F;F432L; T433A; L435S; S438I; D448V; T449F; T464D; M481K; Q488K; S489C;W490R; F505L; A516V; E526V; I537T; P540Q; 139/140 Q3T; G14A; Q34K; A43G;L71M; Q83E; Q111T; L141V; L149W; +++++ F174I; A209P; W240K; A245G;F246Y; L248V; F277M; A288I; E307C; K326C; D341E; M373L; V383G; N386S;V388K; M390I; M400I; T415A; L426F; F432L; T433A; L435S; S438I; D448V;T449F; T464D; M481K; Q488K; S489C; W490R; K499G; F505L; A516V; E526V;I537T; P540Q; 141/142 Q3T; G14A; Q34K; A43G; L71M; Q83E; Q111T; L141V;L149W; +++++ F174I; A209P; W240K; A245G; F246Y; L248V; F277M; A288I;E307C; K326C; D341E; M373L; V383G; N386S; V388K; M390I; M400I; T415A;L426F; F432L; T433A; L435S; S438I; D448V; T449F; T464D; M481K; Q488K;S489C; W490R; F505L; A516V; E526V; I537T; P540Q; 143/144 Q3T; G14A;Q34K; K38E; A43G; L71M; Q83E; Q111T; L141V; +++++ L149W; F174I; A209P;W240K; A245G; F246Y; L248I; Q272R; F277M; A288I; E307C; K326C; D341E;M373L; V383G; N386S; V388K; M390I; M400I; T415A; L426F; F432L; T433A;L435S; S438I; D448V; T449F; T464D; M481K; Q488K; S489C; W490R; F505L;E512N; A516V; E526V; I537T; P540Q; A541G; 145/146 Q3T; G14A; Q34K; A43G;L71M; Q83E; Q111T; L141V; L149W; +++++ F174I; A209P; W240F; A245G;F246Y; L248V; F277M; A288I; E307C; K326C; D341E; M373L; V383G; N386S;V388K; M390I; M400I; T415A; L426F; F432L; T433A; L435S; S438I; D448V;T449F; T464D; E480N; M481K; Q488K; S489C; W490R; F505L; A516V; E526V;I537T; P540Q; 147/148 Q3T; G14A; Q34K; A43G; L71M; Q83E; Q111T; L141V;L149W; +++++ F174I; A209P; W240K; A245G; F246Y; L248V; F277M; A288I;E307C; K326C; D341E; M373L; A377G; V383G; N386S; V388K; M390I; M400I;T415A; L426F; F432L; T433A; L435S; S438I; D448V; T449F; T464D; M481K;Q488K; S489C; W490R; F505L; A516V; E526V; I537T; P540Q; 149/150 Q3T;G14A; Q34K; A43G; L71M; Q83E; Q111T; L141V; A146T; +++++ L149W; F174I;A194E; A209P; W240K; A245G; F246Y; L248V; F277M; A288I; E307C; K326C;D341E; M373L; A377G; V383G; N386S; V388K; M390I; M400I; T415A; L426F;F432L; T433A; L435S; S438I; D448V; T449F; T464D; E480N; M481K; Q488K;S489C; W490R; F505L; E512N; A516V; E526V; I537T; P540Q; 151/152 Q3T;G14A; Q34K; A43G; L71M; Q83E; Q111T; L141V; A146T; +++++ L149W; F174I;A194E; A209P; W240K; A245G; F246Y; L248V; F277M; A288I; E307C; P312Q;K326C; D341E; M373L; A377G; V383G; N386S; V388K; M390I; M400I; T415A;L426F; F432L; T433A; L435S; S438I; D448V; T449F; T464D; M481K; Q488K;S489C; W490R; F505L; E512N; A516V; E526V; I537T; P540Q; 153/154 Q3T;G14A; Q34K; A43G; L71M; Q83E; Q111T; L141V; A146T; +++++ L149W; F174I;A194E; A209P; W240K; A245G; F246Y; L248V; F277M; A288I; E307C; P312Q;K326C; D341E; M373L; A377G; V383G; N386S; V388K; M390I; M400I; T415A;L426F; F432L; T433A; L435S; S438I; D448V; T449F; T464D; M481K; Q488K;S489C; W490R; F505L; A516V; E526V; I537T; P540Q; 155/156 Q3T; G14A;Q34K; A43G; L71M; Q111T; L141V; L149W; F174I; +++++ A209P; W240K; F246Y;L248V; F277M; A288I; E307R; K326C; D341E; V383G; N386S; V388K; M390I;M400I; T415A; L426F; F432L; T433A; L435S; S438I; D448V; T449F; M481K;Q488K; S489C; W490R; F505L; A516V; E526V; I537T; P540Q; 157/158 Q3T;G14A; Q34K; A43G; L71M; Q111T; L141V; L149W; F174I; +++++ A209P; W240K;F246Y; L248V; F277M; A288I; E307R; K326C; D341E; V383G; N386S; V388K;M390I; M400I; T415A; L426F; F432L; T433A; L435S; S438I; D448V; T449F;T464D; M481K; Q488K; S489C; W490R; F505L; A516V; E526V; I537T; P540Q; += >1.5 fold ++ = ≧10 fold +++ = ≧100 fold ++++ = ≧1000 fold +++++ =≧10000 fold

In some embodiments, the polypeptide capable of converting compound (1)to compound (2a) in enantiomeric excess at a rate that is greater than1.5 fold the rate of SEQ ID NO:2 can comprise an amino acid sequencethat is at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identical to a reference sequenceselected from the group consisting of SEQ ID NO: 4, 6, 160, 162, 164,166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192,194, 196, 198, 200, 202, 204, 206, and 208 with the proviso that thepolypeptide amino acid sequence comprises any one of the set of residuedifferences as compared to SEQ ID NO:2 present in the polypeptidesequences represented by SEQ ID NO: 4, 6, 160, 162, 164, 166, 168, 170,172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198,200, 202, 204, 206, or 208 as provided in Table 2A or 2B. In someembodiments, the polypeptides can have additionally 1-2, 1-3, 1-4, 1-5,1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20,1-22, 1-24, 1-26, 1-30, 1-35, 1-40 residue differences at other aminoacid residue positions as compared to the reference sequence. In someembodiments, the number of differences can be 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35, and 40 residuedifferences at other residue positions. The residue difference at theseother positions can include conservative changes or non-conservativechanges. In some embodiments, the residue differences can compriseconservative substitutions and non-conservative substitutions ascompared to the reference sequence. In some embodiments, the residuedifferences are conservative substitutions.

In some embodiments, the polypeptide capable of converting compound (1)to compound (2b) in enantiomeric excess can comprise an amino acidsequence that is at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a referencesequence of SEQ ID NO: 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30,32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66,68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100,102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128,130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156,158, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234,236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262,264, and 266.

In some embodiments, the polypeptide capable of converting compound (1)to compound (2b) in enantiomeric excess can comprise an amino acidsequence that is at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a referencesequence selected from the group consisting of SEQ ID NO: 8, 10, 12, 14,16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50,52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86,88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116,118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144,146, 148, 150, 152, 154, 156, 158, 210, 212, 214, 216, 218, 220, 222,224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250,252, 254, 256, 258, 260, 262, 264, and 266, with the proviso that thepolypeptide amino acid sequence comprises any one of the set of residuedifferences, as compared to SEQ ID NO:2, present in any one of thepolypeptide sequences represented by SEQ ID NO:8 to SEQ ID NO:158 inTable 2A or SEQ ID NO: 210 to SEQ ID NO: 266 in Table 2B. In someembodiments, the polypeptides can have additionally 1-2, 1-3, 1-4, 1-5,1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20,1-22, 1-24, 1-26, 1-30, 1-35, 1-40 residue differences at other aminoacid residue positions as compared to the reference sequence. In someembodiments, the number of differences can be 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35, and 40 residuedifferences at other residue positions. The residue difference at theseother positions can include conservative changes or non-conservativechanges. In some embodiments, the residue differences can compriseconservative substitutions and non-conservative substitutions ascompared to the reference sequence. In some embodiments, the residuedifferences are conservative substitutions.

Table 2B below provides 53 exemplary non-naturally occurringmonooxygenase polypeptides capable of converting compound (1) tocompound (2) that differ by one amino acid residue difference fromreference polypeptides of SEQ ID NO: 4, 16, 32, 52, 56, 58, and 76. Oddnumbered SEQ ID NOs refer to the nucleotide sequence encoding the aminoacid sequence provided by the even numbered SEQ ID NOs. The residuedifferences in Table 2B are provided based on comparison to thewild-type reference sequence of SEQ ID NO:2, as well as the referencepolypeptides of SEQ ID NO: 4, 16, 32, 52, 56, 58, and 76.

The Activity FIOP and enantioselectivity (% ee) of the 53 exemplarynon-naturally occurring monooxygenase polypeptides of Table 2B incarrying out the biocatalytic conversion of the substrate compound (1)(pyrmetazole) to the product compound (2) ((R)- or (S)-omeprazole) weredetermined that following general HTP assay conditions: 5 g/Lpyrmetazole substrate, 10 μL of lysate of the engineered CHMOpolypeptide, 1 g/L KRED of SEQ ID NO: 268, 0.5 g/L NADP, in a solutionof 50 mM potassium phosphate buffer, 10% (v/v) IPA, pH 9.0, 25° C.reaction temperature and 24 h reaction time (with 400 rpm stirring).Further details of the HTP assay methods are described in the Examples.

TABLE 2B Residue Difference Activity (relative to FLOP SEQ parent(relative % ee ID NO: reference Residue Differences to SEQ ID of S-(nt/aa) sequence) (relative to SEQ ID NO: 2) NO: 2) isomer 159/160L426H + L426H; F432A; T433A; L435S; W490R; + −82.4 SEQ ID NO: 4 161/162L426Q + L426Q; F432A; T433A; L435S; W490R; + −96.1 SEQ ID NO: 4 163/164A432H + F432H; T433A; L435S; W490R; + −69.1 SEQ ID NO: 4 165/166 A432K +F432K; T433A; L435S; W490R; + −98.7 SEQ ID NO: 4 167/168 A432T + F432T;T433A; L435S; W490R; + −95.9 SEQ ID NO: 4 169/170 A432D + F432D; T433A;L435S; W490R; + −100.0 SEQ ID NO: 4 171/172 A432C + F432C; T433A; L435S;W490R; + −69.4 SEQ ID NO: 4 173/174 A432I + F432I; T433A; L435S;W490R; + −98.8 SEQ ID NO: 4 175/176 A432L + F432L; T433A; L435S; W490R;++ −94.1 SEQ ID NO: 4 177/178 A432Y + F432Y; T433A; L435S; W490R; +−90.8 SEQ ID NO: 4 179/180 A433F + F432A; T433F; L435S; W490R; + −69.1SEQ ID NO: 4 181/182 A433K + F432A; T433K; L435S; W490R; + −68.8 SEQ IDNO: 4 183/184 A433W + F432A; T433W; L435S; W490R; + −90.1 SEQ ID NO: 4185/186 R490E + F432A; T433A; L435S; W490E; ++ −83.7 SEQ ID NO: 4187/188 R490P + F432A; T433A; L435S; W490P; ++ −96.4 SEQ ID NO: 4189/190 F277V + F277V; F432A; T433A; L435S; W490R; + −100.0 SEQ ID NO: 4191/192 R278H + R278H; F432A; T433A; L435S; W490R; + −100.0 SEQ ID NO: 4193/194 F279Y + F279Y; F432A; T433A; L435S; W490R; + −88.7 SEQ ID NO: 4195/196 M280W + M280W; F432A; T433A; L435S; W490R; ++ −99.9 SEQ ID NO: 4197/198 F281H + F281H; F432A; T433A; L435S; W490R; ++ −88.1 SEQ ID NO: 4199/200 K326A + K326A; F432A; T433A; L435S; W490R; ++ −100.0 SEQ ID NO:4 201/202 K326S + K326S; F432A; T433A; L435S; W490R; ++ −92.9 SEQ ID NO:4 203/204 K326L + K326L; F432A; T433A; L435S; W490R; ++ −99.5 SEQ ID NO:4 205/206 K326D + K326D; F432A; T433A; L435S; W490R; + −100.0 SEQ ID NO:4 207/208 S489P + F432A; T433A; L435S; S489P; W490R; ++ −95.3 SEQ ID NO:4 209/210 L149M + Q3T; L149M; K326C; N386S; F432L; T433A; L435S; ++ SEQID S438I; D448V; W490R; NO: 16 211/212 F277L + Q3T; F277L; K326C; N386S;F432L; T433A; L435S; ++ SEQ ID S438I; D448V; W490R; NO: 16 213/214L144A + Q3T; L144A; K326C; N386S; F432L; T433A; L435S; ++ SEQ ID S438I;D448V; W490R; NO: 16 215/216 R278N + Q3T; R278N; K326C; N386S; F432L;T433A; L435S; ++ SEQ ID S438I; D448V; W490R; NO: 16 217/218 L143V + Q3T;L143V; K326C; N386S; F432L; T433A; L435S; ++ SEQ ID S438I; D448V; W490R;NO: 16 219/220 L143N + Q3T; L143N; K326C; N386S; F432L; T433A; L435S; ++SEQ ID S438I; D448V; W490R; NO: 16 221/222 A433L + Q3T; K326C; N386S;F432L; T433L; L435S; S438I; ++ SEQ ID D448V; W490R; NO: 16 223/224L149F + Q3T; Q34K; L149F; A209P; W240K; A288I; K326C; +++ SEQ ID N386S;T415A; F432L; T433A; L435S; S438I; D448V; NO: 32 W490R; A516V; I537T;225/226 C330G + Q3T; Q34K; A209P; W240K; A288I; K326C; C330G; +++ SEQ IDN386S; T415A; F432L; T433A; L435S; S438I; D448V; NO: 32 W490R; A516V;I537T; 227/228 L426F + Q3T; G14A; Q34K; L141V; A146V; F174L; A209P; +++SEQ ID W240K; A245G; F277L; A288I; K326C; C330G; N386S; NO: 56 T415A;L426F; F432L; T433A; L435S; S438I; D448V; W490R; A516V; I537T; 229/230E282S + Q3T; Q34K; L141V; A209P; W240K; E282S; A288I; ++++ SEQ ID K326C;N386S; T415A; F432L; T433A; L435S; S438I; NO: 52 D448V; S489C; W490R;A516V; I537T; 231/232 F505L + Q3T; G14A; Q34K; L141V; A209P; W240K;A288I; +++++ SEQ ID K326C; N386S; T415A; L426F; F432L; T433A; L435S; NO:58 S438I; D448V; S489C; W490R; F505L; A516V; I537T; 233/234 L149W + Q3T;G14A; Q34K; L141V; L149W; A209P; W240K; +++++ SEQ ID A288I; K326C;N386S; T415A; L426F; F432L; T433A; NO: 58 L435S; S438I; D448V; S489C;W490R; A516V; I537T; 235/236 F246E + Q3T; G14A; Q34K; Q111T; L141V;A209P; W240K; ++++ SEQ ID F246E; A288I; E307R; K326C; N386S; V388K;T415A; NO: 76 L426F; F432L; T433A; L435S; S438I; D448V; M481K; S489C;W490R; F505L; A516V; I537T; 237/238 F246H + Q3T; G14A; Q34K; Q111T;L141V; A209P; W240K; ++++ 14.7 SEQ ID F246H; A288I; E307R; K326C; N386S;V388K; T415A; NO: 76 L426F; F432L; T433A; L435S; S438I; D448V; M481K;S489C; W490R; F505L; A516V; I537T; 239/240 F246Q + Q3T; G14A; Q34K;Q111T; L141V; A209P; W240K; ++++ 45.9 SEQ ID F246Q; A288I; E307R; K326C;N386S; V388K; T415A; NO: 76 L426F; F432L; T433A; L435S; S438I; D448V;M481K; S489C; W490R; F505L; A516V; I537T; 241/242 F246S + Q3T; G14A;Q34K; Q111T; L141V; A209P; W240K; +++++ 98.4 SEQ ID F246S; A288I; E307R;K326C; N386S; V388K; T415A; NO: 76 L426F; F432L; T433A; L435S; S438I;D448V; M481K; S489C; W490R; F505L; A516V; I537T; 243/244 F277Q + Q3T;G14A; Q34K; Q111T; L141V; A209P; W240K; +++++ 97.7 SEQ ID F277Q; A288I;E307R; K326C; N386S; V388K; T415A; NO: 76 L426F; F432L; T433A; L435S;S438I; D448V; M481K; S489C; W490R; F505L; A516V; I537T; 245/246 F246W +Q3T; G14A; Q34K; Q111T; L141V; A209P; W240K; +++ 80.8 SEQ ID F246W;A288I; E307R; K326C; N386S; V388K; T415A; NO: 76 L426F; F432L; T433A;L435S; S438I; D448V; M481K; S489C; W490R; F505L; A516V; I537T; 247/248M280G + Q3T; G14A; Q34K; Q111T; L141V; A209P; W240K; +++++ 97.9 SEQ IDM280G; A288I; E307R; K326C; N386S; V388K; T415A; NO: 76 L426F; F432L;T433A; L435S; S438I; D448V; M481K; S489C; W490R; F505L; A516V; I537T;249/250 R278G + Q3T; G14A; Q34K; Q111T; L141V; A209P; W240K; +++++ 98.4SEQ ID R278G; A288I; E307R; K326C; N386S; V388K; T415A; NO: 76 L426F;F432L; T433A; L435S; S438I; D448V; M481K; S489C; W490R; F505L; A516V;I537T; 251/252 M280R + Q3T; G14A; Q34K; Q111T; L141V; A209P; W240K; ++SEQ ID M280R; A288I; E307R; K326C; N386S; V388K; T415A; NO: 76 L426F;F432L; T433A; L435S; S438I; D448V; M481K; S489C; W490R; F505L; A516V;I537T; 253/254 R278S + Q3T; G14A; Q34K; Q111T; L141V; A209P; W240K;+++++ 98.5 SEQ ID R278S; A288I; E307R; K326C; N386S; V388K; T415A; NO:76 L426F; F432L; T433A; L435S; S438I; D448V; M481K; S489C; W490R; F505L;A516V; I537T; 255/256 F281A + Q3T; G14A; Q34K; Q111T; L141V; A209P;W240K; +++++ 98.2 SEQ ID F281A; A288I; E307R; K326C; N386S; V388K;T415A; NO: 76 L426F; F432L; T433A; L435S; S438I; D448V; M481K; S489C;W490R; F505L; A516V; I537T; 257/258 F281S + Q3T; G14A; Q34K; Q111T;L141V; A209P; W240K; +++++ 94.0 SEQ ID F281S; A288I; E307R; K326C;N386S; V388K; T415A; NO: 76 L426F; F432L; T433A; L435S; S438I; D448V;M481K; S489C; W490R; F505L; A516V; I537T; 259/260 Q488K + Q3T; G14A;Q34K; Q111T; L141V; A209P; W240K; +++++ SEQ ID A288I; E307R; K326C;N386S; V388K; T415A; L426F; NO: 76 F432L; T433A; L435S; S438I; D448V;M481K; Q488K; S489C; W490R; F505L; A516V; I537T; 261/262 Q488F + Q3T;G14A; Q34K; Q111T; L141V; A209P; W240K; +++++ 97.1 SEQ ID A288I; E307R;K326C; N386S; V388K; T415A; L426F; NO: 76 F432L; T433A; L435S; S438I;D448V; M481K; Q488F; S489C; W490R; F505L; A516V; I537T; 263/264 Q488L +Q3T; G14A; Q34K; Q111T; L141V; A209P; W240K; +++++ 97.5 SEQ ID A288I;E307R; K326C; N386S; V388K; T415A; L426F; NO: 76 F432L; T433A; L435S;S438I; D448V; M481K; Q488L; S489C; W490R; F505L; A516V; I537T; 265/266F246R + Q3T; G14A; Q34K; Q111T; L141V; A209P; W240K; +++ SEQ ID F246R;A288I; E307R; K326C; N386S; V388K; T415A; NO: 76 L426F; F432L; T433A;L435S; S438I; D448V; M481K; S489C; W490R; F505L; A516V; I537T; + = >1.5fold ++ = ≧10 fold +++ = ≧100 fold ++++ = ≧500 fold +++++ = ≧1000 fold

Accordingly, in some embodiments, the present disclosure provides andengineered polypeptide capable of converting compound (1) to compound(2) can comprise an amino acid sequence that is at least about 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore identical to a reference sequence of SEQ ID NO: 8, 10, 12, 14, 16,18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52,54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88,90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118,120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146,148, 150, 152, 154, 156, or 158, and comprises one or more amino aciddifferences relative to the reference sequence selected from: X143N;X143V; X144A; X149F; X149M; X149W; X246E; X246H; X246Q; X246R; X246S;X246W; X277L; X277Q; X277V; X278G; X278H; X278N; X278S; X279Y; X280G;X280R; X280W; X281A; X281H; X281S; X282S; X326A; X326S; X326L; X326D;X330G; X426F; X426H; X426Q; X432C; X432D; X432H; X4321; X432K; X432L;X432T; X432Y; X433F; X433K; X433L; X433W; X488K; X488F; X488L; X489P;X490E; X490P; and X505L (i.e., the amino acid differences of Table 2B).In some embodiments, the engineered polypeptide comprises a sequence atleast about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more identical to a sequence of any one of SEQ IDNO: 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184,186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212,214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240,242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, or 266. Insome embodiments, the engineered polypeptide is capable of convertingcompound (1) to either the (R)- or the (S)-enantiomer of compound (2) inenantiomeric excess.

In some embodiments, the present disclosure provides an engineeredpolypeptide is capable of converting a structurally similar analog ofthe substrate pyrmetazole (e.g., a compound of structural formula (I))to either the (R)- or the (S)-enantiomer of the corresponding analogprazole compound (e.g., compound of structural formula (II)) inenantiomeric excess, which comprises an amino acid sequence that is atleast about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more identical to a reference sequence of SEQ IDNO: 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40,42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76,78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108,110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136,138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164,166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190; 192,194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220,222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248,250, 252, 254, 256, 258, 260, 262, 264, or 266. In some embodiments, theengineered polypeptide is capable of converting an pyrmetazole analogsubstrate to an omeprazole analog compound selected from: (R) or(S)-lansoprazole, (R) or (S)-tenatoprazole, (R) or (S)-rabeprazole, (R)or (S)-pantoprazole, (R) or (S)-ilaprazole, (R) or (S)-leminoprazole,(R) or (S)-saviprazole, and (R) or (S)-TY-11345.

In some embodiments, the present disclosure provides an engineeredpolypeptide capable of converting the pyrmetazole analog substrate5-(difluoromethoxy)-2-((3,4-dimethoxypyridin-2-yl)methylthio)-1H-benzo[d]imidazoleto the omeprazole analog compound, (S)-pantoprazole in enantiomericexcess. In some embodiments, the polypeptide capable of producing(S)-pantoprazole in enantiomeric excess comprises an amino acid sequenceselected from the group consisting of SEQ ID NO: 70, 72, 76, 78, 80, 82,84, 86, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116,118, 120, 122, 126, 128, 130, 228, 232, 244, 248, 256, 258, 260, 262,and 264.

In some embodiments, the present disclosure provides an engineeredpolypeptide capable of converting the pyrmetazole analog substrate5-methoxy-2-((4-methoxy-3,5-dimethylpyridin-2-yl)methylthio)-1H-imidazo[4,5-b]pyridineto the omeprazole analog compound, (S)-tenatoprazole, in enantiomericexcess. In some embodiments, the polypeptide capable of producing(S)-tenatoprazole comprises an amino acid sequence selected from thegroup consisting of SEQ ID NO: 66, 102, 104, 114, 122, 124, 128, 232,238, 244, 260, 262, and 264.

In some embodiments, the present disclosure provides an engineeredpolypeptide capable of converting the pyrmetazole analog substrate2-((4-(3-methoxypropoxy)-3-methylpyridin-2-yl)methylthio)-1H-benzo[d]imidazoleto the omeprazole analog compound, (S)-rabeprazole, in enantiomericexcess. in some embodiments, the polypeptide capable of producing(S)-rabeprazole comprises an amino acid sequence selected from the groupconsisting of SEQ ID NO: 62, 76, 84, 86, 124, 238, 240, 250, 258, 262,and 264.

The present disclosure also contemplates engineered CHMO polypeptidescapable of converting compound (1) to compound (2), where thepolypeptides comprise an amino acid sequence comprising mutations basedon locations or regions in the structure of the parent polypeptide.Accordingly, referring to Table 3, a variant of a parent polypeptide(e.g., SEQ ID NO: 2) can include an amino acid substitution at aparticular residue at a location in the structure of the parentpolypeptide as identified in Table 3. Exemplary substitutions at each ofthe relevant locations are also identified in Table 3.

TABLE 3 Structural Locations Useful for Engineered CHMO PolypeptidesCorresponding Position in SEQ ID NO: 2 Structural Location X3 SurfaceX14 Buried - close to FAD X15 Buried - FAD-binding X22 Buried (nonactive site) X32 Surface X34 Surface X39 Partially Buried (FAD-Bindingsite) X43 Partially Buried - Near FAD X44 Buried - FAD-Binding X59Surface (near active site) X71 Partially Buried (non-active site) X74Surface X83 Surface X92 Surface X107 Surface X111 Surface X113 Buried(non active site) X114 Surface X123 Surface X141 Buried - FAD-BindingX146 Partially Buried X149 Surface X154 Surface X155 Surface X161Surface X174 Buried (non active site) X176 Surface X194 Surface X195Buried (non active site) X199 Buried (non active site) X201 Surface X209Surface - close to NADP X240 Surface X244 Active Site X245 Active SiteX246 Active Site X248 Surface X277 Active Site X288 Surface X307 SurfaceX326 Partially Buried (Active Site) X329 Buried (active site main chain)X330 Buried (active site main chain) X341 Surface X354 Surface X367Surface X368 Surface X383 Active Site X386 Surface (FAD-Binding site)X388 Surface X390 Buried - FAD-Binding X400 Buried (non-active site)X408 Partially Buried (non active site) X415 Buried (non active site)X426 Active Site X428 Buried (active site main chain) X432 Active SiteX433 Active Site X435 Active Site X438 Active Site X448 Surface X449Surface X451 Buried (non active site) X454 Surface X459 Surface X475Buried (non active site) X481 Surface X488 Surface X489 PartiallyBuried - Active Site X490 Active Site X499 Surface X505 Active Site X507Partially Buried (near active site) X516 Surface X526 Surface X532Surface X537 Surface X540 Surface

In some embodiments, the polypeptides can comprise deletions of theengineered monooxygenase polypeptides described herein. Thus, for eachand every embodiment of the polypeptides of the disclosure, thedeletions can comprise one or more amino acids, 2 or more amino acids, 3or more amino acids, 4 or more amino acids, 5 or more amino acids, 6 ormore amino acids, 8 or more amino acids, 10 or more amino acids, 15 ormore amino acids, or 20 or more amino acids, up to 10% of the totalnumber of amino acids, up to 10% of the total number of amino acids, upto 20% of the total number of amino acids of the polypeptides, as longas the functional activity of the monooxygenase polypeptide as describedherein is maintained. In some embodiments, the functional activity iswith respect to the conversion of compound (1) to compound (2) at a rategreater than 1.5 fold the rate of the monooxygenase of SEQ ID NO:2. Insome embodiments, the functional activity of the polypeptide is withrespect to the conversion of compound (1) to compound (2b) inenantiomeric excess. In some embodiments, the deletions can comprise,1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15,1-16, 1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35, or 1-40 amino acidresidues. In some embodiments, the number of deletions can be 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35, or40 amino acids. In some embodiments, the deletions can comprisedeletions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18,or 20 amino acid residues.

In some embodiments, the polypeptides can comprise fragments of theengineered polypeptides described herein. In some embodiments, thefragments can have about 80%, 90%, 95%, 98%, and 99% of the full-lengthpolypeptide, e.g., the polypeptide of SEQ ID NO:8, as long as thefunctional activity of the polypeptide is maintained. In someembodiments, the functional activity is with respect to the conversionof compound (1) to compound (2) at a rate greater than 1.5 fold the rateof the monooxygenase of SEQ ID NO: 2. In some embodiments, thefunctional activity of the polypeptide with respect to the conversion ofcompound (1) to compound (2b) in enantiomeric excess.

In some embodiments, the polypeptides of the disclosure can be in theform of fusion polypeptides in which the engineered polypeptides arefused to other polypeptides, such as, by way of example and notlimitation, antibody tags (e.g., myc epitope), purifications sequences(e.g., His tags for binding to metals), and cell localization signals(e.g., secretion signals). Thus, the engineered polypeptides describedherein can be used with or without fusions to other polypeptides.

As will be understood by the skilled artisan, the polypeptides describedherein are not restricted to the genetically encoded amino acids. Inaddition to the genetically encoded amino acids, the polypeptidesdescribed herein may be comprised, either in whole or in part, ofnaturally-occurring and/or synthetic non-encoded amino acids. Certaincommonly encountered non-encoded amino acids of which the polypeptidesdescribed herein may be comprised include, but are not limited to: theD-enantiomers of the genetically-encoded amino acids;2,3-diaminopropionic acid (Dpr); α-aminoisobutyric acid (Aib);ε-aminohexanoic acid (Aha); δ-aminovaleric acid (Ava); N-methylglycineor sarcosine (MeGly or Sar); ornithine (Orn); citrulline (Cit);t-butylalanine (Bua); t-butylglycine (Bug); N-methylisoleucine (MeIle);phenylglycine (Phg); cyclohexylalanine (Cha); norleucine (Nle);naphthylalanine (NaI); 2-chlorophenylalanine (Ocf);3-chlorophenylalanine (Mcf); 4-chlorophenylalanine (Pcf);2-fluorophenylalanine (Off); 3-fluorophenylalanine (Mff);4-fluorophenylalanine (Pff); 2-bromophenylalanine (Obf);3-bromophenylalanine (MW); 4-bromophenylalanine (Pbf);2-methylphenylalanine (Omf); 3-methylphenylalanine (Mmf);4-methylphenylalanine (Pmf); 2-nitrophenylalanine (Onf);3-nitrophenylalanine (Mnf); 4-nitrophenylalanine (Pnf);2-cyanophenylalanine (Ocf); 3-cyanophenylalanine (Mcf);4-cyanophenylalanine (Pcf); 2-trifluoromethylphenylalanine (Otf);3-trifluoromethylphenylalanine (Mtf); 4-trifluoromethylphenylalanine(Ptf); 4-aminophenylalanine (Paf); 4-iodophenylalanine (Pif);4-aminomethylphenylalanine (Pamf); 2,4-dichlorophenylalanine (Opef);3,4-dichlorophenylalanine (Mpcf); 2,4-difluorophenylalanine (Opfl);3,4-difluorophenylalanine (Mpff); pyrid-2-ylalanine (2pAla);pyrid-3-ylalanine (3pAla); pyrid-4-ylalanine (4pAla); naphth-1-ylalanine(1nAla); naphth-2-ylalanine (2nAla); thiazolylalanine (taAla);benzothienylalanine (bAla); thienylalanine (tAla); furylalanine (fAla);homophenylalanine (hPhe); homotyrosine (hTyr); homotryptophan (hTrp);pentafluorophenylalanine (5ff); styrylkalanine (sAla); authrylalanine(aAla); 3,3-diphenylalanine (Dfa); 3-amino-5-phenypentanoic acid (Afp);penicillamine (Pen); 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid(Tic); β-2-thienylalanine (Thi); methionine sulfoxide (Mso);N(w)-nitroarginine (nArg); homolysine (hLys);phosphonomethylphenylalanine (pmPhe); phosphoserine (pSer);phosphothreonine (pThr); homoaspartic acid (hAsp); homoglutanic acid(hGlu); 1-aminocyclopent-(2 or 3)-ene-4 carboxylic acid; pipecolic acid(PA), azetidine-3-carboxylic acid (ACA);1-aminocyclopentane-3-carboxylic acid; allylglycine (aOly);propargylglycine (pgGly); homoalanine (hAla); norvaline (nVal);homoleucine (hLeu), homovaline (hVal); homoisolencine (hlle);homoarginine (hArg); N-acetyl lysine (AcLys); 2,4-diaminobutyric acid(Dbu); 2,3-diaminobutyric acid (Dab); N-methylvaline (MeVal);homocysteine (hCys); homoserine (hSer); hydroxyproline (Hyp) andhomoproline (hPro). Additional non-encoded amino acids of which thepolypeptides described herein may be comprised will be apparent to thoseof skill in the art (see, e.g., the various amino acids provided inFasman, 1989, CRC Practical Handbook of Biochemistry and MolecularBiology, CRC Press, Boca Raton, Fla., at pp. 3-70 and the referencescited therein, all of which are incorporated by reference). These aminoacids may be in either the L- or D-configuration.

Those of skill in the art will recognize that amino acids or residuesbearing side chain protecting groups may also comprise the polypeptidesdescribed herein. Non-limiting examples of such protected amino acids,which in this case belong to the aromatic category, include (protectinggroups listed in parentheses), but are not limited to: Arg(tos),Cys(methylbenzyl), Cys (nitropyridinesulfenyl), Glu(δ-benzylester),Gln(xanthyl), Asn(N-δ-xanthyl), His(bom), His(benzyl), His(tos),Lys(fmoc), Lys(tos), Ser(O-benzyl), Thr (O-benzyl) and Tyr(O-benzyl).

Non-encoding amino acids that are conformationally constrained of whichthe polypeptides described herein may be composed include, but are notlimited to, N-methyl amino acids (L-configuration); 1-aminocyclopent-(2or 3)-ene-4-carboxylic acid; pipecolic acid; azetidine-3-carboxylicacid; homoproline (hPro); and 1-aminocyclopentane-3-carboxylic acid.

In some embodiments, the polypeptides can be present in whole cellstransformed with gene(s) encoding the engineered monooxygenase enzyme,or as cell extracts, lysates, isolated polypeptide, or substantiallypurified, in a variety of different forms, including solid (e.g.,lyophilized, spray-dried, and the like) or semisolid (e.g., a crudepaste).

In some embodiments, the polypeptide described herein can be provided inthe form of kits. The enzymes in the kits may be present individually oras a plurality of enzymes. The kits can further include reagents forcarrying out the enzymatic reactions, substrates for assessing theactivity of enzymes, as well as reagents for detecting the products. Thekits can also include reagent dispensers and instructions for use of thekits.

In some embodiments, the polypeptides can be provided on a physicalsubstrate. In some embodiments, the polypeptides can be provided in theform of an array in which the polypeptides are arranged in positionallydistinct locations. The array can be used to test a variety of arylalkyl sulfides for conversion by the polypeptides. “Substrate,”“support,” “solid support,” “solid carrier,” or “resin” in the contextof arrays refer to any solid phase material. Substrate also encompassesterms such as “solid phase,” “surface,” and/or “membrane.” A solidsupport can be composed of organic polymers such as polystyrene,polyethylene, polypropylene, polyfluoroethylene, polyethyleneoxy, andpolyacrylamide, as well as co-polymers and grafts thereof. A solidsupport can also be inorganic, such as glass, silica, controlled poreglass (CPG), reverse phase silica or metal, such as gold or platinum.The configuration of a substrate can be in the form of beads, spheres,particles, granules, a gel, a membrane or a surface. Surfaces can beplanar, substantially planar, or non-planar. Solid supports can beporous or non-porous, and can have swelling or non-swellingcharacteristics. A solid support can be configured in the form of awell, depression, or other container, vessel, feature, or location. Aplurality of supports can be configured on an array at variouslocations, addressable for robotic delivery of reagents, or by detectionmethods and/or instruments.

In certain embodiments, the kits of the present disclosure includearrays comprising a plurality of different engineered monooxygenasepolypeptides at different addressable position, wherein the differentpolypeptides are different variants of a reference sequence each havingat least one different improved enzyme property. Such arrays comprisinga plurality of engineered polypeptides and methods of their use aredescribed in, e.g., WO2009/008908A2.

5.3 POLYNUCLEOTIDES, EXPRESSION VECTORS, AND HOST CELLS

In another aspect, the present disclosure provides polynucleotidesencoding the polypeptides described herein. The polynucleotides may beoperatively linked to one or more heterologous regulatory sequences thatcontrol gene expression to create a recombinant polynucleotide capableof expressing the monooxygenase polypeptide. Expression constructscontaining a heterologous polynucleotide encoding the engineeredmonooxygenase can be introduced into appropriate host cells to expressthe corresponding polypeptide.

It is to be understood that the availability of a polypeptide amino acidsequence provides a description of all the polynucleotides capable ofencoding the subject polypeptide because of the knowledge of the codonscorresponding to the various amino acids. Thus, having identified aparticular amino acid sequence, those skilled in the art could make anynumber of different nucleic acids by simply modifying the sequence ofone or more codons in a way which does not change the amino acidsequence of the protein. In this regard, the present disclosurespecifically contemplates each and every possible variation ofpolynucleotides that could be made by selecting combinations based onthe possible codon choices, and all such variations are to be consideredspecifically disclosed for any polypeptide disclosed herein, includingthe amino acid sequences presented in Tables 2A and 2B.

In some embodiments, the polynucleotides can be selected and/orengineered to comprise codons that are preferably selected to fit thehost cell in which the protein is being produced. For example, preferredcodons used in bacteria are used to express the gene in bacteria;preferred codons used in yeast are used for expression in yeast; andpreferred codons used in mammals are used for expression in mammaliancells. Since not all codons need to be replaced to optimize the codonusage of the monooxygenases (e.g., because the natural sequence can havepreferred codons and because use of preferred codons may not be requiredfor all amino acid residues), codon optimized polynucleotides encodingthe monooxygenase polypeptides may contain preferred codons at about40%, 50%, 60%, 70%, 80%, or greater than 90% of codon positions of thefull length coding region.

In some embodiments, the polynucleotide encodes a polypeptide comprisingan amino acid sequence that has at least about 80%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or moresequence identity to an amino acid sequence selected from the groupconsisting of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26,28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62,64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98,100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126,128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154,156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182,184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210,212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238,240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, and266, wherein the polypeptide is capable of converting compound (1) tocompound (2) at a rate that is greater than 1.5 fold the rate of themonooxygenase of SEQ ID NO:2.

In some embodiments, the polynucleotide encodes a polypeptide comprisingan amino acid sequence that has at least about 80%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or moresequence identity to an amino acid sequence selected from the groupconsisting of SEQ ID NO: 4 and 6, wherein the polypeptide is capable ofconverting compound (1) to compound (2a) in enantiomeric excess and at arate that is greater than 1.5 fold the rate of the monooxygenase of SEQID NO:2. In some embodiments, the polynucleotide encodes a polypeptidecapable of converting compound (1) to compound (2a) in enantiomericexcess and comprises an amino acid sequence that is at least about 80%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identical to a reference sequence based on SEQ ID NO: 4, 6, 160,162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188,190, 192, 194, 196, 198, 200, 202, 204, 206, or 208, with the provisothat the amino acid sequence comprises any one of the set of residuedifferences as compared to SEQ ID NO:2 as present in the polypeptidesequences of SEQ ID NO: 4, 6, 160, 162, 164, 166, 168, 170, 172, 174,176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202,204, 206, or 208, as provided in Tables 2A or 2B.

In some embodiments, the polynucleotide encodes a polypeptide comprisingan amino acid sequence that has at least about 80%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or moresequence identity to an amino acid sequence selected from the groupconsisting of SEQ ID NO: 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30,32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66,68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100,102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128,130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156,158, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234,236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262,264, and 266, wherein the polypeptide is capable of converting compound(1) to compound (2b) in enantiomeric excess.

In some embodiments, the polynucleotide encodes a polypeptide capable ofconverting compound (1) to compound (2b) in enantiomeric excess andcomprises an amino acid sequence that is at least about 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to a reference sequence selected from the group consisting ofSEQ ID NO: 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36,38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72,74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106,108, 110, 112, 114, 116, 118, 120, 122; 124, 126, 128, 130, 132, 134,136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 210, 212,214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240,242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, and 266,with the proviso that the amino acid sequence comprises any one of theset of residue differences as compared to SEQ ID NO:2 as present in anyone of the polypeptide sequences of SEQ ID NO:8 to SEQ ID NO:158 asprovided in Table 2A or SEQ ID NO: 210 to SEQ ID NO: 266 as provided inTable 2B.

In some embodiments, the polynucleotides encoding the polypeptides areselected from the group consisting of SEQ ID NO: 3, 5, 7, 9, 11, 13, 15,17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51,53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87,89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117,119, 121, 123, 125, 126, 127, 129, 131, 133, 135, 137, 139, 141, 143,145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171,173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199,201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227,229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255,257, 259, 261, 263, and 265.

In some embodiments, the polynucleotides are capable of hybridizingunder highly stringent conditions to a polynucleotide comprising SEQ IDNO: 3, 5, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181,183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, or 207, or acomplement thereof, where the highly stringently hybridizingpolynucleotides encode a monooxygenase polypeptide capable of convertingcompound (1) to compound (2a) in enantiomeric excess at a rate greaterthan 1.5 fold the rate of the monooxygenase of SEQ ID NO:2.

In some embodiments, the polynucleotides are capable of hybridizingunder highly stringent conditions to a polynucleotide selected from thegroup consisting of SEQ ID NO: 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27,29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63,65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99,101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127,129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155,157, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233,235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261,263, and 265, or a complement thereof, where the highly stringentlyhybridizing polynucleotides encode a monooxygenase polypeptide capableof converting compound (1) to compound (2b) in enantiomeric excess.

In some embodiments, the polynucleotides encode the polypeptidesdescribed herein but have about 80% or more sequence identity, about80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% or more sequence identity at the nucleotide level to areference polynucleotide encoding the engineered monooxygenasesdescribed herein. In some embodiments, the reference polynucleotide isselected from the group consisting of SEQ ID NO: 3, 5, 7, 9, 11, 13, 15,17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51,53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87,89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117,119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145,147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173,175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201,203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229,231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257,259, 261, 263, and 265.

An isolated polynucleotide encoding a polypeptide of the disclosure maybe manipulated in a variety of ways to provide for expression of thepolypeptide. In some embodiments, the polynucleotides encoding thepolypeptides can be provided as expression vectors where one or morecontrol sequences is present to regulate the expression of thepolynucleotides. Manipulation of the isolated polynucleotide prior toits insertion into a vector may be desirable or necessary depending onthe expression vector. The techniques for modifying polynucleotides andnucleic acid sequences utilizing recombinant DNA methods are well knownin the art. Guidance is provided in Sambrook et al., 2001, MolecularCloning: A Laboratory Manual, 3rd Ed., Cold Spring Harbor LaboratoryPress; and Current Protocols in Molecular Biology, Ausubel. F. ed.,Greene Pub. Associates, 1998, updates to 2009, the disclosures of whichare incorporated herein by reference.

In some embodiments, the control sequences include among others,promoters, leader sequences, polyadenylation sequences, propeptidesequences, signal peptide sequences, and transcription terminators.Suitable promoters can be selected based on the host cells used.Exemplary bacterial promoters include E. coli lac operon, E. coli trpoperon, bacteriophage λ, Streptomyces coelicolor agarase gene (dagA),Bacillus subtilis levansucrase gene (sacB), Bacillus licheniformisalpha-amylase gene (amyL), beta-lactamase gene, and tac promoter.Exemplary promoters for filamentous fungal host cells, include promotersobtained from the genes for Aspergillus oryzae TAKA amylase, Rhizomucormiehei aspartic proteinase, Aspergillus niger neutral alpha-amylase,Aspergillus niger acid stable alpha-amylase, Aspergillus niger orAspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase,Aspergillus oryzae alkaline protease, Aspergillus oryzae triosephosphate isomerase, Aspergillus nidulans acetamidase, and Fusariumoxysporum trypsin-like protease, and mutant, truncated, and hybridpromoters thereof. Exemplary yeast cell promoters can be from the genesfor Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP), andSaccharomyces cerevisiae 3-phosphoglycerate kinase.

In some embodiments, the control sequence may also be a signal peptidecoding region that codes for an amino acid sequence linked to the aminoterminus of a polypeptide and directs the encoded polypeptide into thecell's secretory pathway. The signal sequence typically depends on thetype of host cell being used to express the polypeptide. Effectivesignal peptide coding regions for bacterial host cells are the signalpeptide coding regions obtained from the genes for Bacillus NC1B 11837maltogenic amylase, Bacillus stearothermophilus alpha-amylase, Bacilluslicheniformis subtilisin, Bacillus licheniformis beta-lactamase,Bacillus stearothermophilus neutral proteases (nprT, nprS, nprM), andBacillus subtilis prsA. Exemplary signal peptide coding regions forfilamentous fungal host cells can be the signal peptide coding regionsobtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillusniger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor mieheiaspartic proteinase, Humicola insolens cellulase, and Humicolalanuginosa lipase. Useful signal peptides for yeast host cells can befrom the genes for Saccharomyces cerevisiae alpha-factor andSaccharomyces cerevisiae invertase.

Other control sequences, such as leader sequences, polyadenylationsequences, and transcription terminator sequences can use thoseavailable in the art (see Sambrook, supra, and Current Protocols inMolecular Biology, supra).

In another aspect, the present disclosure is also directed to arecombinant expression vector comprising a polynucleotide encoding anengineered monooxygenase polypeptide or a variant thereof, and one ormore expression regulating regions such as a promoter and a terminator,a replication origin, etc., depending on the type of hosts into whichthey are to be introduced. The recombinant expression vector may be anyvector (e.g., a plasmid, cosmid, or virus), which can be convenientlysubjected to recombinant DNA procedures and can bring about theexpression of the polynucleotide sequence. The choice of the vector willtypically depend on the compatibility of the vector with the host cellinto which the vector is to be introduced. The vectors may be linear orclosed circular plasmids.

The expression vector may be an autonomously replicating vector, i.e., avector that exists as an extrachromosomal entity, the replication ofwhich is independent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one which, when introduced into thehost cell, is integrated into the genome and replicated together withthe chromosome(s) into which it has been integrated. The expressionvector preferably contains one or more selectable markers, which permiteasy selection of transformed cells. A selectable marker is a gene theproduct of which provides for biocide or viral resistance, resistance toheavy metals, prototrophy to auxotrophs, resistance to chemical agents(e.g., antibiotics) and the like.

In another aspect, the present disclosure provides a host cellcomprising a polynucleotide encoding an engineered monooxygenasepolypeptide of the present disclosure, the polynucleotide beingoperatively linked to one or more control sequences for expression ofthe monooxygenase polypeptide in the host cell. Host cells for use inexpressing the monooxygenase polypeptides encoded by the expressionvectors of the present invention are well known in the art and includebut are not limited to, bacterial cells, such as E. coli, Lactobacillus,Streptomyces and Salmonella typhimurium cells; fungal cells, such asyeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9cells; animal cells such as CHO, COS, BHK, 293, and Bowes melanomacells; and plant cells. Exemplary host cells are Escherichia coli BL21and W3110.

Appropriate culture mediums and growth conditions for theabove-described host cells are well known in the art. Polynucleotidesfor expression of the monooxygenase may be introduced into host cells byvarious methods known in the art (e.g., electroporation, biolisticparticle bombardment, liposome mediated transfection, calcium chloridetransfection, and protoplast fusion).

In the embodiments herein, the monooxygenase polypeptides andnucleotides encoding such polypeptides can be prepared using methodscommonly used by those skilled in the art. As noted above, thenaturally-occurring amino acid sequence and corresponding polynucleotideencoding the cyclohexanone monooxygenase enzyme of Acinetobacter spNCIMB9871 (represented herein as SEQ ID NO:2) is described in Chen etal., 1988, J. Bacteriol. 170 (2), 781-789 and Genbank Accession No.BAA86293.1 GI:6277322. In some embodiments, the parent polynucleotidesequence is codon optimized to enhance expression of the monooxygenasein a specified host cell.

The engineered monooxygenases can be obtained by subjecting thepolynucleotide encoding the naturally occurring cyclohexanonemonooxygenase to mutagenesis and/or directed evolution methods (e.g.,Stemmer, 1994, Proc Natl Acad Sci USA 91:10747-10751; WO 95/22625; WO97/0078; WO 97/35966; WO 98/27230; WO 00/42651; WO 01/75767 and U.S.Pat. No. 6,537,746; each of which is hereby incorporated by referenceherein).

Other directed evolution procedures that can be used include, amongothers, staggered extension process (StEP), in vitro recombination (Zhaoet al., 1998, Nat. Biotechnol. 16:258-261), mutagenic PCR (Caldwell etal., 1994, PCR Methods Appl. 3:S136-S140), and cassette mutagenesis(Black et al., 1996, Proc Natl Acad Sci USA 93:3525-3529). Mutagenesisand directed evolution techniques useful for the purposes herein arealso described in the following references: Ling, et al., 1997, Anal.Biochem. 254(2):157-78; Dale et al., 1996, Methods Mol. Biol. 57:369-74;Smith, 1985, Ann. Rev. Genet. 19:423-462; Botstein et al., 1985, Science229:1193-1201; Carter, 1986, “Site-directed mutagenesis,” Biochem. J.237:1-7; Kramer et al., 1984, Cell 38:879-887; Wells et al., 1985, Gene34:315-323; Minshull et al., 1999, Curr Opin Chem Biol 3:284-290;Christians et al., 1999, Nature Biotech 17:259-264; Crameri et al.,1998, Nature 391:288-291; Crameri et al., 1997, Nature Biotech15:436-438; Zhang et al., 1997, Proc Natl Acad Sci USA 94:45-4-4509;Crameri et al., 1996, Nature Biotech 14:315-319; and Stemmer, 1994,Nature 370:389-391. All publications are incorporated herein byreference.

In some embodiments, the clones obtained following mutagenesis treatmentare screened for monooxygenases having a desired enzyme property.Measuring monooxygenase enzyme activity from the expression librariescan be performed using the standard techniques, such as separation ofthe product (e.g., by HPLC) and detection of the product by measuring UVabsorbance of the separated substrate and products and/or by detectionusing tandem mass spectroscopy (e.g., MS/MS). Clones containing apolynucleotide encoding the desired polypeptides are then isolated,sequenced to identify the nucleotide sequence changes (if any), and usedto express the enzyme in a host cell. Exemplary assays for monooxygenaseactivity are provided in Example 2.

Where the sequence of the polypeptide is known, the polynucleotidesencoding the enzyme can be prepared by standard solid-phase methods,according to known synthetic methods, e.g., the phosphoramidite methoddescribed by Beaucage et al., 1981, Tet Lett 22:1859-69, or the methoddescribed by Matthes et al., 1984, EMBO J. 3:801-05. In someembodiments, fragments of up to about 100 bases can be individuallysynthesized, then joined (e.g., by enzymatic or chemical litigationmethods, or polymerase mediated methods) to form any desired continuoussequence.

The polypeptides can be expressed in appropriate cells, and recoveredfrom the host cells and or the culture medium using any one or more ofthe well known techniques used for protein purification, including,among others, lysozyme treatment, sonication, filtration, salting-out,ultra-centrifugation, and chromatography. Chromatographic techniques forisolation of the monooxygenase polypeptide include, among others,reverse phase chromatography high performance liquid chromatography, ionexchange chromatography, gel electrophoresis, and affinitychromatography.

Conditions for purifying a particular enzyme will depend, in part, onfactors such as net charge, hydrophobicity, hydrophilicity, molecularweight, molecular shape, etc., and will be apparent to those havingskill in the art. In some embodiments, the engineered monooxygenases canbe expressed as fusion proteins with purification tags, such as His-tagshaving affinity for metals, or antibody tags for binding to antibodies,e.g., myc epitope tag.

The polypeptide of the disclosure can be prepared in the form of crudeextracts, lyophizates, powders, isolated preparations, and substantiallypure preparations, as further described below.

5.4 METHODS OF USE

In a further aspect, the monooxygenase polypeptides of the disclosurecan be used in a process for conversion of various aryl-alkyl sulfidesubstrates to the corresponding sulfoxide. While the monooxygenasepolypeptides herein are described with respect to the conversion of5-methoxy-2-((4-methoxy-3,5-dimethylpyridin-2-yl)methylthio)-1H-benzo[d]imidazole(compound (1)) to5-methoxy-2-((4-methoxy-3,5-dimethylpyridin-2-yl)methylsulfinyl)-1H-benzo[d]imidazole(compound (2)), the engineered monooxygenase polypeptides can be appliedto the conversion of other prazole compounds structurally similar topyrmetazole. Accordingly in some embodiments, the monooxygenasepolypeptides of the disclosure can be used in a process for theconversion of the compound of structural Formula (I) to the product ofFormula (II):

wherein, Ar¹ is an optionally substituted aryl or heteroaryl ring; R isH, a lower alkyl, a heteroalkyl, or forms a 5 to 8 membered cycloalkyl,heteroalkyl, aryl or heteroaryl fused ring with a ring carbon of Ar¹;and Ar² is an optionally substituted cycloalkyl, heterocycloalkyl, aryl,or heteroaryl ring fused to the imidazole ring.

In some embodiments, Ar¹ is an optionally substituted phenyl or pyridyl.In some embodiments, Ar¹ can have 1 to 5 substitutions, preferably 1, 2,or 3 substitutions. Substitutions in Ar¹ can be a substituted orunsubstituted: lower alkyl, lower alkoxy, amino or alkylamino group. Insome embodiments, the substituted alkyl is a haloalkyl.

In some embodiments, Ar² is selected from an optionally substitutedthienyl, phenyl or pyridyl. In some embodiments Ar² can have 1 to 5substitutions, preferably 1, 2 or 3 substitutions. Substitutions in Ar¹can be a substituted or unsubstituted: lower alkyl, lower alkoxy, or a 5to 7 membered heterocycloalkyl, aryl or heteroaryl ring.

Prazole compounds that can be prepared by use of the monooxygenasesdescribed herein include, by way of example and not limitation, thoseshown in Table 4 below.

TABLE 4 Compound Name Prazole Compound Structure Esomeprazole (or(S)-omeprazole)

(R)-Omeprazole

(R)- or (S)-Lansoprazole

(R)- or (S)-Tenatoprazole

(R)- or (S)-Rabeprazole

(R)- or (S)-Pantoprazole

(R)- or (S)-Ilaprazole

(R)- or (S)-Leminoprazole

(R)- or (S)-Saviprazole

(R)- or (S)-TY-11345

In some embodiments, the process can comprise contacting or incubatingthe compound of formula (I) above with an engineered CHMO polypeptidedescribed herein in presence of an electron donor under suitablereaction conditions to convert the compound of formula (I) to theproduct compound of formula (II). Suitable reaction conditions include asource of molecular oxygen (O₂), and the electron donor can be cofactorNADPH or NADH. In some embodiments, the O₂ can be O₂ dissolved in areaction solution. The enantioselectivity of the process can bedetermined by measuring the amount of (R) and (S) products formed in thereaction. Exemplary polypeptides for use in the process can be apolypeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26,28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62,64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98,100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126,128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154,156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182,184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210,212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238,240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, and266, or an engineered CHMO polypeptide that comprises an amino acidsequence having the set of amino acid differences relative to SEQ ID NO:2 that are present in any one of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18,20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54,56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90,92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120,122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148,150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176,178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204,206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232,234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260,262, 264, or 266.

In some embodiments, the engineered CHMO polypeptide useful in theprocess can comprise an amino acid sequence at least 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moreidentical to a reference sequence selected from the group consisting ofSEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34,36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70,72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104,106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132,134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160,162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188,190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216,218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244,246, 248, 250, 252, 254, 256, 258, 260, 262, 264, or 266, and optionallyfurther comprising an amino acid sequence having the set of amino aciddifferences relative to SEQ ID NO: 2 that are listed in Table 2A or 2Bfor any one of 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68,70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102,104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130,132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158,160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186,188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214,216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242,244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, or 266.

As described herein, in some embodiments, the monooxygenase polypeptidesof the disclosure can be used in a process for the conversion of5-methoxy-2-((4-methoxy-3,5-dimethylpyridin-2-yl)methylthio)-1H-benzo[d]imidazole(“pyrmetazole” or “compound (1)”) to5-methoxy-2-((4-methoxy-3,5-dimethylpyridin-2-yl)methylsulfinyl)-1H-benzo[d]imidazole(“R- and S-omeprazole” or “compound (2)”). In some embodiments, theprocess comprises contacting or incubating compound (1) with apolypeptide described herein in presence of a an electron donor, e.g., acofactor, under suitable reaction conditions to convert compound (1) tocompound (2).

In some embodiments, the monooxygenase polypeptides can be used in theconversion of compound (1) to compound (2a) in enantiomeric excess. Insome embodiments, the process comprises contacting or incubatingcompound (1) with a R-enantioselective polypeptide described herein inpresence of an electron donor, e.g., a cofactor, under suitable reactionconditions to convert the compound (1) to compound (2a) in enantiomericexcess. Exemplary polypeptides useful for the process can comprise anamino acid sequence selected from the group consisting of SEQ ID NO: 4,6, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186,188, 190, 192, 194, 196, 198, 200, 202, 204, 206, and 208, or anengineered CHMO polypeptide that comprises an amino acid sequence havingthe set of amino acid differences relative to SEQ ID NO: 2 that arepresent in any one of SEQ ID NO: 4, 6, 160, 162, 164, 166, 168, 170,172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198,200, 202, 204, 206, or 208.

In some embodiments, the monooxygenase polypeptides can be used in theconversion of compound (1) to compound (2b) in enantiomeric excess. Insome embodiments, the process comprises contacting or incubating thecompound (1) with a S-enantioselective polypeptide described herein inpresence of an electron donor, e.g., a cofactor, under suitable reactionconditions to convert the compound (1) to compound (2b) in enantiomericexcess. Exemplary polypeptides useful for the process can comprise anamino acid sequence selected from SEQ ID NO: 8, 10, 12, 14, 16, 18, 20,22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56,58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92,94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122,124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150,152, 154, 156, 158, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228,230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256,258, 260, 262, 264, and 266, or an engineered CHMO polypeptide thatcomprises an amino acid sequence having the set of amino aciddifferences relative to SEQ ID NO: 2 that are present in any one of SEQID NO: 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38,40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74,76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108,110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136,138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 210, 212, 214,216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242,244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, or 266.

In some embodiments, the process comprises contacting or incubating thecompound (1) with a S-enantioselective polypeptide described herein inpresence of an electron donor, e.g., a cofactor, under suitable reactionconditions to convert the compound (1) to compound (2b) in at least 90%enantiomeric excess. Exemplary polypeptides useful for the process cancomprise an amino acid sequence selected from SEQ ID NO: 10, 12, 14, 16,18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52,54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88,90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118,120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146,148, 150, 152, 154, 156, 158, 242, 244, 248, 250, 254, 256, 258, 262,and 264, or an engineered CHMO polypeptide that comprises an amino acidsequence having the set of amino acid differences relative to SEQ ID NO:2 that are present in any one of SEQ ID NO: 10, 12, 14, 16, 18, 20, 22,24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58,60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94,96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124,126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152,154, 156, 158, 242, 244, 248, 250, 254, 256, 258, 262, or 264.

In some embodiments, the process comprises contacting or incubating thecompound (1) with a polypeptide described herein in presence of anelectron donor, e.g., a cofactor, under suitable reaction conditions toconvert the compound (1) to compound (2b) in at least 99% enantiomericexcess. Exemplary polypeptides useful for this process can comprise anamino acid sequence selected from SEQ ID NO: 16, 20, 22, 24, 26, 28, 30,32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66,68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100,102, 104, 106, 108, 110, 112, 114, 116, 116, 120, 122, 124, 126, 128,130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156,and 158, or an engineered CHMO polypeptide that comprises an amino acidsequence having the set of amino acid differences relative to SEQ ID NO:2 that are present in any one of SEQ ID NO: 16, 20, 22, 24, 26, 28, 30,32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66,68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100,102, 104, 106, 108, 110, 112, 114, 116, 116, 120, 122, 124, 126, 128,130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, or158.

In some embodiments, the monooxygenase polypeptides can be used in thepreparation of an omeprazole analog compound of structural formula (II)in enantiomeric excess, wherein the compound of structural formula (II)is selected from: (R) or (S)-lansoprazole, (R) or (S)-tenatoprazole, (R)or (S)-rabeprazole, (R) or (S)-pantoprazole, (R) or (S)-ilaprazole, (R)or (S)-leminoprazole, (R) or (S)-saviprazole, and (R) or (S)-TY-11345.In such embodiments, process comprises contacting or incubating asulfide precursor of structural formula (I) for the omeprazole analogcompound (i.e., the corresponding pyrmetazole substrate analog compound)with a polypeptide described herein in presence of an electron donor(e.g., a cofactor) under suitable reaction conditions to convert thesulfide precursor compound of formula (I) to the omeprazole analogcompound of structural formula (II).

In some embodiments, this process for preparing an omeprazole analogcompound can be carried out wherein the sulfide precursor compound offormula (I) is5-(difluoromethoxy)-2-((3,4-dimethoxypyridin-2-yl)methylthio)-1H-benzo[d]imidazoleand the compound of formula (II) is (5)-pantoprazole which is producedin enantiomeric excess. In such embodiments, the process can be carriedout wherein the polypeptide comprises an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 70, 72, 76, 78, 80, 82, 84, 86,90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118,120, 122, 126, 128, 130, 228, 232, 244, 248, 256, 258, 260, 262, and264, or in which the polypeptide comprises an amino acid sequence havingthe set of amino acid differences relative to SEQ ID NO: 2 that arepresent in any one of ID NO: 70, 72, 76, 78, 80, 82, 84, 86, 90, 92, 94,96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 126,128, 130, 228, 232, 244, 248, 256, 258, 260, 262, or 264.

In some embodiments, this process for preparing an omeprazole analogcompound can be carried out wherein the sulfide precursor compound offormula (I) is5-methoxy-2-((4-methoxy-3,5-dimethylpyridin-2-yl)methylthio)-1H-imidazo[4,5-b]pyridineand the compound of formula (II) is (S)-tenatoprazole, which is producedin enantiomeric excess. In such embodiments, the process can be carriedout wherein the polypeptide comprises an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 66, 102, 104, 114, 122, 124,128, 232, 238, 244, 260, 262, and 264, or in which the polypeptidecomprises an amino acid sequence having the set of amino aciddifferences relative to SEQ ID NO: 2 that are present in any one of SEQID NO: 66, 102, 104, 114, 122, 124, 128, 232, 238, 244, 260, 262, or264.

In some embodiments, this process for preparing an omeprazole analogcompound can be carried out wherein the sulfide precursor compound offormula (I) is244-(3-methoxypropoxy)-3-methylpyridin-2-yl)methylthio)-1H-benzo[d]imidazoleand the compound of formula (II) is (S)-rabeprazole, which is producedin enantiomeric excess. In such embodiments, the process can be carriedout wherein the polypeptide comprises an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 62, 76, 84, 86, 124, 238, 240,250, 258, 262, and 264, or in which the polypeptide comprises an aminoacid sequence having the set of amino acid differences relative to SEQID NO: 2 that are present in any one of SEQ ID NO: 62, 76, 84, 86, 124,238, 240, 250, 258, 262, or 264.

In some embodiments, this process for preparing an omeprazole analogcompound can be carried out wherein the sulfide precursor compound offormula (I) is2-((3-methyl-4-(2,2,2-trifluoroethoxy)pyridin-2-yl)methylthio)-1H-benzo[d]imidazoleand the compound of formula (II) is (R)-lansoprazole which is producedin enantiomeric excess. In such embodiments, the process can be carriedout wherein the polypeptide comprises an amino acid sequence of SEQ IDNO: 6, or in which the polypeptide comprises an amino acid sequencehaving the set of amino acid differences relative to SEQ ID NO: 2 thatare present in SEQ ID NO: 6.

In some embodiments, the process is carried out at a reaction conditiontemperature of 10° C. to 50° C., and in particular 25° C. to 40° C. Thetemperature can be chosen to maximize the reaction rate at highertemperatures while maintaining the activity of the enzyme for sufficientduration for efficient conversion of the substrate to the product. Wherehigher temperatures are used, polypeptides with increasedthermostability can be selected to carry out the process.

In some embodiments, the reaction condition comprises a pH of about 8.5to a pH of about 10. In some embodiments, the reaction condition is a pHof about 8.5 to about 9. This slightly basic condition limits thedegradation of the product compound (2) that occurs below about pH 8.5.During the course of the reaction, the pH of the reaction mixture maychange. The pH of the reaction mixture may be maintained at a desired pHor within a desired pH range by the addition of an acid or a base duringthe course of the reaction. Alternatively, the pH may be controlled byusing an aqueous solvent that comprises a buffer. Suitable buffers tomaintain desired pH ranges are known in the art and include, forexample, phosphate buffer, triethanolamine buffer, and the like.Combinations of buffering and acid or base addition may also be used. Insome embodiments, the reaction condition comprises a phosphate bufferconcentration of about 5 to 50 mM. At the lower phosphate bufferconcentrations, NADPH is shown to have greater stability.

As noted above, the process described herein consumes molecular oxygen,i.e., O₂, where an oxygen atom is transferred to a sulfide to yield thesulfoxide. In some embodiments, the O₂ is dissolved in the reactionsolution. Dissolved O₂ can be increased by direct sparging of O₂ gasinto the reaction solution (e.g., U.S. Pat. No. 6,478,964), and/or byincreasing the partial pressure of O₂ to pressures higher than theatmospheric pressure. In some embodiments, oxygenation of the reactionsolution is done by a bubble-free process. For example, oxygen masstransfer across PTFE membrane for bubble free aeration is described inSchneider et al., 1995, Enzyme and Microbial Technology 17(9):839-847and EPO 172478, incorporated herein by reference.

Generally, a cofactor is used in the reduction reaction. The cofactoroperates in combination with the polypeptides of the disclosure in theprocess. Suitable cofactors include, but are not limited to, NADP⁺(nicotinamide adenine dinucleotide phosphate), NADPH (the reduced formof NADP⁺), NAD⁺ (nicotinamide adenine dinucleotide) and NADH (thereduced form of NAD⁺). Generally, the reduced form of the cofactor isadded to the reaction mixture. The reduced NAD(P)H form can beoptionally regenerated from the oxidized NAD(P)⁺ form using a cofactorregeneration system. In some embodiments of the process, a cofactorrecycling system is used to regenerate cofactor NADPH/NADH formNADP+/NAD+ produced in the reaction.

In some embodiments of the process, an optional cofactor recyclingsystem can be used to regenerate cofactor NADPH/NADH form NADP+/NAD+produced in the reaction. A cofactor regeneration system refers to a setof reactants that participate in a reaction that reduces the oxidizedform of the cofactor (e.g., NADP⁺ to NADPH). Cofactors oxidized by thepolypeptide reduction of the keto substrate are regenerated in reducedform by the cofactor regeneration system. Cofactor regeneration systemscomprise a stoichiometric reductant that is a source of reducinghydrogen equivalents and is capable of reducing the oxidized form of thecofactor. The cofactor regeneration system may further comprise acatalyst, for example an enzyme catalyst that catalyzes the reduction ofthe oxidized form of the cofactor by the reductant. Cofactorregeneration systems to regenerate NADH or NADPH from NAD⁺ or NADP⁺,respectively, are known in the art and may be used in the methodsdescribed herein.

Suitable exemplary cofactor regeneration systems that may be employedinclude, but are not limited to, glucose and glucose dehydrogenase;formate and formate dehydrogenase; glucose-6-phosphate andglucose-6-phosphate dehydrogenase; an alcohol (e.g., isopropanol) and aketoreductase/alcohol dehydrogenase; phosphite and phosphitedehydrogenase, molecular hydrogen and hydrogenase; and the like. Thesesystems may be used in combination with either NADP⁺/NADPH or NAD⁺/NADHas the cofactor. Electrochemical regeneration using hydrogenase may alsobe used as a cofactor regeneration system. See, e.g., U.S. Pat. Nos.5,538,867 and 6,495,023, both of which are incorporated herein byreference. Chemical cofactor regeneration systems comprising a metalcatalyst and a reducing agent (for example, molecular hydrogen orformate) are also suitable. See, e.g., PCT publication WO 2000/053731,which is incorporated herein by reference.

In some embodiments, the cofactor recycling system can comprise glucosedehydrogenase (GDH), which is a NAD⁺ or NADP⁺-dependent enzyme thatcatalyzes the conversion of D-glucose and NAD⁺ or NADP⁺ to gluconic acidand NADH or NADPH, respectively. Glucose dehydrogenases suitable for usein the practice of the processes described herein include both naturallyoccurring glucose dehydrogenases, as well as non-naturally occurringglucose dehydrogenases. Naturally occurring glucose dehydrogenaseencoding genes have been reported in the literature, e.g., the Bacillussubtilis 61297 GDH gene, B. cereus ATCC 14579 and B. megaterium.Non-naturally occurring glucose dehydrogenases generated using, forexample, mutagenesis, directed evolution, and the like are provided inPCT publication WO 2005/018579, and US publication Nos. 2005/0095619 and2005/0153417. All of these sequences are incorporated herein byreference.

In some embodiments, the co-factor regenerating system can comprise aformate dehydrogenase, which is a NAD⁺ or NADV-dependent enzyme thatcatalyzes the conversion of formate and NAD⁺ or NADP⁺ to carbon dioxideand NADH or NADPH, respectively. Formate dehydrogenases that aresuitable for use as cofactor regenerating systems in the monooxygenasereactions described herein include naturally occurring and non-naturallyoccurring formate dehydrogenases. Suitable formate dehydrogenases aredescribed in PCT publication WO 2005/018579. Formate may be provided inthe form of a salt, typically an alkali or ammonium salt (for example,HCO₂Na, KHCO₂NH₄, and the like), in the form of formic acid, typicallyaqueous formic acid, or mixtures thereof. A base or buffer may be usedto provide the desired pH.

In some embodiments, the co-factor regenerating system can comprise aphosphite dehydrogenase, which catalyzes the conversion of phosphite andNAD″ or NADP″ to a phosphate and NADH or NADPH, respectively. Phosphitedehydrogenases that are suitable for use as cofactor regeneratingsystems in the processes described herein include naturally occurringand non-naturally occurring phosphite dehydrogenases. Naturallyoccurring phosphite dehydrogenases include those from, Pseudomonasstutzeri and Alcaligenes faecalis, and non-naturally occurring phosphitedehydrogenases include engineered phosphite dehydrogenases derivedtherefrom. Phosphite dehydrogenases are described in Johannes et al.,2005, Applied and Environmental Microbiology 71(10):5728-5734; Woodyeret al., 2003, Biochemistry 42 (40):11604-11614; Vrtis et al., 2002,Angewandte Chemie 41(17):3257-3259; Johannes et al., 2006, Biotechnologyand Bioengineering Volume 96(1):18-26; and McLachlan et al., 2008,Biotechnology and Bioengineering 99(2):268-274.

In some embodiments, the co-factor regenerating system can comprise analcohol dehydrogenase or ketoreductase, which is an NAD⁺ orNADP⁺-dependent enzyme that catalyzes the conversion of an alcohol andNAD⁺ or NADP⁺ to an aldehyde or ketone and NADH or NADPH, respectively.Alcohol dehydrogenases and ketoreductases that are suitable for use ascofactor regenerating systems in the processes described herein includenaturally occurring and non-naturally occurring alcohol dehydrogenasesand ketoreductases. Naturally occurring alcohol dehydrogenases includeknown alcohol dehydrogenase/ketoreductase from, among others,Thermoanerobium brockii, Rhodococcus erythropolis, Saccharomycescerevisiae, Lactobacillus kefiri, Lactobacillus minor, and Lactobacillusbrevis, and non-naturally occurring alcohol dehydrogenase/ketoreductaseinclude engineered alcohol dehydrogenase/ketoreductase derivedtherefrom. In some embodiments, non-naturally occurring alcoholdehydrogenase/ketoreductases engineered for thermo- and solventstability can be used. Such alcohol dehydrogenases/ketoreductases aredescribed in patent publications US 20080318295; 20090093031;20090191605; US 20090155863; and US 20090162909; all of which areincorporated by reference herein.

Suitable alcohols include lower secondary alkanols and aryl-alkylcarbinols. Examples of lower secondary alcohols include isopropanol,2-butanol, 3-methyl-2-butanol, 2-pentanol, 3-pentanol,3,3-dimethyl-2-butanol, and the like. In one embodiment, the secondaryalcohol is isopropanol. Suitable aryl-alkyl carbinols includeunsubstituted and substituted 1-arylethanols.

In some embodiments where the cofactor recycling system produces avolatile product, such as acetone from isopropanol, the volatile productcan be removed by sparging the reaction solution with a non-reactive gasor by applying a vacuum to lower the reaction pressure and removing thevolatile present in the gas phase. A non-reactive gas is any gas thatdoes not react with the reaction components. Various non-reactive gasesinclude nitrogen and noble gases (e.g., inert gases). In someembodiments, the non-reactive gas is nitrogen gas. For example, acetoneformed by oxidation of isopropanol can be removed by sparging withnitrogen gas or applying a vacuum to the reaction solution and removingthe acetone from the gas phase by an acetone trap, such as a condenseror other cold trap.

In the embodiments herein, the polypeptides for carrying out theconversion of pyrmetazole to esomeprazole and any enzymes comprising theoptional cofactor regeneration system, may be added to the reactionmixture in the form of the purified enzymes, whole cells transformedwith gene(s) encoding the enzymes, and/or cell extracts and/or lysatesof such cells. The gene(s) encoding the polypeptides disclosed hereinand the optional cofactor regeneration enzymes can be transformed intohost cells separately or together into the same host cell. Whole cellstransformed with gene(s) encoding the engineered ketoreductase enzymeand/or the optional cofactor regeneration enzymes, or cell extractsand/or lysates thereof, may be employed in a variety of different forms,including solid (e.g., lyophilized, spray-dried, and the like) orsemisolid (e.g., a crude paste).

The processes described herein are generally carried out in an aqueoussolvent (e.g., water, buffer, and salts). In some embodiments, aqueoussolvents, including water and/or co-solvent systems, are used.Co-solvents can reduce the formation of aggregates which can affect therate and scalability of the process. At substrate loading of 75 g/L orhigher, the use of a co-solvent is desirable. Suitable co-solventsinclude: MeOH, EtOH, isopropanol (IPA), acetone, toluene, MeCN, methyltert-butyl ether (MTBE), N-methyl-2-pyrrolidone (NMP), dimethylacetamide(DMAc), dimethylformamide (DMF), propylene glycol, polyethylene glycol(PEG), tetramethylurea, N-ethylpyrollidinone, tetraglyme,1,3-Dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone (DMPU), DMIU,hexamethylphosphoramide (HMPA) and dimethylsulfoxide (DMSO). Choice ofco-solvent can be based on evaluating a combination of factorsincluding: compound solubility, compound stability, reaction/processsafety, toxicity, allowable level of solvent in the product (e.g., anAPI product); the effectiveness of the co-solvent in preventingagglomeration of the product, and stability of the monooxygenase to theco-solvent. NMP is a particularly suitable co-solvent for reactions withsubstrate loading of 100 g/L.

The order of addition of reactants is not critical. The reactants may beadded together at the same time to a solvent (e.g., monophasic solvent,biphasic aqueous co-solvent system, and the like), or alternatively,some of the reactants may be added separately, and some together atdifferent time points.

In the processes herein, the reaction is generally allowed to proceeduntil essentially complete, or near complete, conversion of compound (1)with a polypeptide described herein under suitable reaction conditionsto convert the compound (1) to compound (2b) is obtained. Conversion ofsubstrate to product can be monitored using known methods by detectingsubstrate and/or product. Suitable methods include gas chromatography,HPLC, and the like.

6. EXAMPLES

Various features and embodiments of the disclosure are illustrated inthe following representative examples, which are intended to beillustrative, and not limiting.

Example 1 Wild-Type Cyclohexanone Monooxygenase (CHMO) Gene Acquisitionand Construction of Expression Vectors

The gene encoding the wild type cyclohexanone monooxygenase (CHMO) fromAcinetobacter sp NCIMB9871 (SEQ ID NO: 2) was designed for expression inE. coli using standard codon optimization to yield the nucleotidesequence of SEQ ID NO: 1. (Standard codon-optimization software isreviewed in, e.g., “OPTIMIZER: a web server for optimizing the codonusage of DNA sequences,” Puigbo et al., Nucleic Acids Res. 2007 July;35(Web Server issue): W126-31. Epub 2007 Apr. 16.) The optimized genewas synthesized using oligonucleotides composed of 42 nucleotides andcloned into expression vector pCK110900 (which is depicted as FIG. 3 inUS Patent Application Publication 20060195947, which is herebyincorporated by reference herein) under the control of a lac promoter.The pCK110900 expression vector also contained the P15a origin ofreplication and the chloramphenicol resistance gene. The resultingplasmid was transformed into E. coli W3110 using standard methods.

Directed evolution of the codon-optimized wild-type CHMO gene wascarried out through multiple rounds resulting in variant polynucleotidesencoding engineered CHMO polypeptides having improved enzyme properties.These variant polynucleotides were cloned into vector pCK110900 forexpression in E. coli W3110 according to the same procedures describedabove for the wild type gene. The variant CHMO nucleotide and amino acidsequences resulting from directed evolution are listed in the SequenceListing incorporated by reference herein. The amino acid residuedifferences and altered enzyme properties of these engineered CHMOpolypeptides are summarized in Tables 2A and 2B (above) and Tables 6, 7,13, 16, and 19 of the Examples below.

Example 2 Assay Procedures for Engineered CHMO Polypeptides

(a) High-Throughput Activity Assay

This example illustrates a high throughput spectrophotometric assay in96-well plate format that is used to assay relative activity ofengineered CHMO polypeptides (as in Table 2A). This assay can also beused for real-time monitoring of bioprocesses using these polypeptides.

The substrate, pyrmetazole (6 g/L), ketoreductase of SEQ ID NO: 268(cofactor recycling enzyme) (1 g/L) and cofactor (0.1 g/L) weredissolved in 300 μL volume of buffer (pH 9.0) with 8% isopropanol (IPA),2% acetone and 15% N-methyl-2-pyrrolidone (NMP) in the wells of a96-well plate. Cell lysate from a directed evolution sample orbioprocess sample containing the engineered CHMO polypeptide (3.3% oftotal reaction volume) was then added to the reaction mixture. Platesalso contained negative control wells (empty vector) which providebackground reaction levels.

The reaction was shaken at ambient temperature for 24 h (or 0.5 h can beused for real-time monitoring of bioprocess) then quenched by dilutionwith 3.33 volumes of acetonitrile and mixed thoroughly to give a total4.3-fold dilution. The quenched mixture is centrifuged at 4000 rpm for10 min. A sample of the quenched mixture was then transferred to anempty 96 well plate. The plate was analyzed with chromatographic methodby high performance liquid chromatography (HPLC) at 250 nm. The assayconditions are summarized in Table 5 below.

TABLE 5 HTP activity assay conditions Chemicals/Reagents AmountSubstrate 6 g/L Cofactor (NADP+) 0.1 g/L Coenzyme (ketoreductase of SEQID NO: 268) 1 g/L Buffer (potassium phosphate, pH 9.0) 75% (v/v)Co-substrate (IPA) 10% (v/v) Co-solvent (acetone) 2% (v/v) Co-solvent(NMP) 15% (v/v) Cell lysate Volume 3.3% Reaction Volume 300 μL ReactionTemperature Ambient

The percentage conversion of the substrate pyrmetazole to esomeprazoleproduct was calculated based on substrate and product peak area obtainedfrom HPLC chromatography as follows:

Percent Conversion=Product peak*Relative response factor/(Substrate peakarea*Response factor+Product peak*Response factor)*100%.

(b) Thermal Stability Assay.

The substrate, pyrmetazole (6 g/L), ketoreductase (KRED) of SEQ ID NO:268 (1 g/L) and NADP⁺ (0.1 g/L) were dissolved in 300 μL volume ofbuffer (pH 9.0) with 8% IPA and 2% acetone in the wells of a 96-wellplate. Cell lysate from a directed evolution or bioprocess samplecontaining the engineered CHMO polypeptide (5% of total reaction volume)was then added to the reaction mixture. Plates also contained negativecontrol wells (empty vector) which provide background reaction levels.

The reaction was shaken at 32° C. for 24 hrs, then quenched by dilutionwith 3.3 volumes of acetonitrile and mixed thoroughly to give a total4.3-fold dilution. The quenched mixture was centrifuged at 4000 rpm for10 min. A sample of the quenched mixture was then transferred to anempty 96 well plate. The plate was analyzed with chromatographic methodby HPLC at 250 nm.

(c) Assay of Sulfone Production

The product, esomeprazole (1 g/L), KRED (1 g/L) and NADP⁺ (0.1 g/L) weredissolved in 300 μL volume of buffer (pH 9.0) with 8% IPA and 2% acetonein the wells of a 96-well plate. Cell lysate from a directed evolutionor bioprocess sample containing the engineered CHMO polypeptide (60% oftotal reaction volume) was then added to the reaction mixture. Platesalso contained negative control wells (empty vector) which providedbackground reaction levels.

The percentage conversion of esomeprazole product to sulfone-byproductof compound (3) was calculated based on esomeprazole andsulfone-byproduct peak areas obtained from HPLC chromatography asfollows: Percent Conversion=Sulfone peak area*Responsefactor/(Esomeprazole peak area*Response factor+Sulfone peakarea*Relative response factor)*100.

(d) Determination of Enantiomeric Excess (% ee)

The substrate, pyrmetazole (2 g/L), KRED (1 g/L) and NADP⁺ (0.1 g/L)were dissolved in 300 μL volume of buffer (pH 9.0) with 8% IPA and 2%acetone in the wells of a 96-well plate. Cell lysate from a directedevolution or bioprocess sample containing the engineered CHMOpolypeptide (60% of total reaction volume) was then added to thereaction mixture. Plates also contained negative controls (empty vector)which provided the background reaction levels.

The percent enantiomeric excess of esomeprazole (or (S)-omeprazole)product was calculated based on (S)-omeprazole and (R)-omeprazole peakareas obtained from HPLC chromatography as follows: Enantiomericexcess=(R)-omeprazole peak area−(S)-omeprazole peak area/((R)-omeprazolepeak area+(S)-omeprazole peak area)×100%. By convention throughout thepresent disclosure, however, the % ee values favoring the(S)-enantiomers have been designated as positive, whereas the % eevalues favoring the (R)-enantiomers have been designated as negative.

Example 3 Production of Engineered CHMO Polypeptide Shake Flask Powders

This example illustrates production and assay of “shake flask powders”of engineered CHMO polypeptides. Shake flask powders includeapproximately 30% total protein and accordingly provide a more purifiedpreparation of an engineered enzyme as compared to the cell lysate usedin the high throughput assay of Example 2.

A single microbial colony of E. coli containing a plasmid encoding anengineered CHMO gene of interest was inoculated into 50 mL Luria Bertanibroth containing 30 μg/mlchloramphenicol and 1% glucose. Cells weregrown overnight (at least 16 hours) in an incubator at 30° C. withshaking at 250 rpm. The culture was diluted into 250 mL Terrific Broth(12 g/L bacto-tryptone, 24 g/L yeast extract, 4 mL/L glycerol, 65 mMpotassium phosphate, pH 7.0, 1 mM MgSO₄) containing 30 μg/mLchloramphenicol, in a 1 L flask to an optical density of 0.2 at 600 nm(OD₆₀₀) and allowed to grow at 30° C. Expression of the CHMO gene wasinduced by addition of isopropyl-β-D-thiogalactoside (IPTG) to a finalconcentration of 1 mM when the OD₆₀₀ of the culture is 0.6 to 0.8, andincubation was then continued overnight (at least 16 hours).

Cells were harvested by centrifugation (5000 rpm, 15 min, 4° C.) and thesupernatant discarded. The cell pellet was resuspended with an equalvolume of cold (4° C.) 25 mM phosphate buffer, pH 9.0, and harvested bycentrifugation as above. The washed cells were resuspended in twovolumes of the cold phosphate buffer and passed through a French Presstwice at 12,000 psi while maintained at 4° C. Cell debris was removed bycentrifugation (9000 rpm, 45 minutes, 4° C.). The clear lysatesupernatant was collected and stored at −20° C. Lyophilization of frozenclear lysate provided a dry shake-flask powder of CHMO polypeptide.Alternatively, the cell pellet (before or after washing) was stored at4° C. or −80° C.

Assays of activity, sulfone-byproduct production, and thermal stabilitywere carried out as describe as in Example 2 except that the followingreagents/conditions were used: 1-10 g/L shake flask powder ofmonooxygenase enzyme; 10-100 g/L pyrmetazole substrate; 1 g/L KREDcoenzyme; 0.2-0.5 g/L NADP+ cofactor; 4% IPA; 96% phosphate buffer (pH9); reaction temperature: room temperature; reaction time: 24 hr. Assayresults for shake flask powder preparations of exemplary engineered CHMOpolypeptides are shown below in Table 6.

TABLE 6 Activity Relative reduction FIOP at 32° C. SEQ ID NO: in %Sulfone- (thermal (nt/aa) Activity FIOP¹ % ee byproduct² stability) 9/10 + 11/12 + 15/16 + 21/22 + 95.6 23/24 + 25/26 + 27/28 + 29/30 +31/32 ++ 96.8 33/34 ++ 35/36 ++ 97.5 37/38 ++ 97.4 39/40 + 96.2 41/42 ++97.4 43/44 + 96.5 45/46 + 47/48 + 49/50 ++ 51/52 ++ 98.7 1.000 53/54 +++0.600 55/56 ++ 57/58 +++ 98.3 0.800 59/60 +++ 98.3 0.800 61/62 +++ 98.30.800 63/64 +++ 98.3 0.800 65/66 ++ 94.4 2.000 67/68 ++ 94.4 2.000 69/70+++ 0.640³ 71/72 +++ 0.560 73/74 +++ 0.720³ 75/76 +++ 98.3 0.224³ 77/78+++ 1.7 79/80 +++ 81/82 +++ 83/84 +++ 98.3 0.067³ 1 85/86 +++ 99.40.090³ 87/88 +++ 99.3 0.045³ 89/90 +++ 99.0 0.022³ 91/92 ++++ 98.60.090³ 93/94 ++ 99.6 0.072³ 95/96 ++++ 99.7 0.027³ 97/98 ++++ 99.70.036³  99/100 ++++ 99.8 0.036³ 101/102 ++++ 99.6 0.090³ 103/104 ++++99.7 0.045³ 105/106 +++ 99.8 0.007³ 107/108 +++++ 99.8 0.007³ 109/110++++ 99.8 0.007³ 111/112 ++++ 99.8 0.007³ 113/114 +++++ 99.7 0.036³115/116 ++++ 99.6 0.014³ 117/118 ++++ 99.8 0.014³ 119/120 ++++ 99.60.007³ 121/122 +++ 99.4 0.014³ 123/124 +++++ 0.007³ 125/126 +++++ 0.007³127/128 +++++ 0.007³ 129/130 +++++ 0.007³ 131/132 ++++++ 0.8⁴ 133/134+++++ 1.5⁴ 135/136 +++++ 137/138 ++++++ 139/140 ++++++ 141/142 +++++143/144 ++++++ 1.2⁵ 145/146 ++++++ 0.7⁵ 147/148 ++++++ 149/150 ++++++1.3⁵ 151/152 ++++++ 1.3⁵ 153/154 ++++++ 1.3 155/156 ++++++ 157/158++++++ ¹Except where noted, the engineered CHMO polypeptide of SEQ IDNO: 10 was used as the “parent” reference sequence for purposes ofcalculating “FIOP” ²Values for relative reduction in % sulfone-byproductof compound (3) are relative to the value measured for the polypeptideof SEQ ID NO: 52 which yielded 72% conversion of pyrmetazole substrateto esomeprazole with only 1.9% sulfone-byproduct formation in a 24-hourreaction using 10 g/L pyrmetazole; 2.5 g/L CHMO polypeptide; 1 g/L KRED;0.5 g/L NADPH; 4% v/v IPA; T = 26-28° C. ⁴Thermostability assay carriedout at 35° C. in solution including 15% NMP. ⁵Thermostability assaycarried out at 35° C. + = ≧1 fold ++ = ≧10 fold +++ = ≧50 fold ++++ =≧100 fold +++++ = ≧200 fold ++++++ = ≧500 fold

Example 4 Fermentation Procedure for Production of Engineered CHMOPolypeptide DSP Powders

This example illustrates production and assay of downstream processed(“DSP”) powders of engineered CHMO polypeptides. DSP powders includeapproximately 80% total protein and accordingly provide a more purifiedpreparation of an engineered enzyme as compared to the cell lysate usedin the high throughput assay of Example 2.

Bench-scale fermentations of engineered CHMO polypeptides were carriedout at 37° C. in batch phase and at 30° C. during expression phase in anaerated, agitated 15 L fermentor using 6.0 L of growth medium (0.88 g/Lammonium sulfate, 0.98 g/L of sodium citrate; 12.5 g/L of dipotassiumhydrogen phosphate trihydrate, 6.25 g/L of potassium dihydrogenphosphate, 3.33 g/L of Tastone-154 yeast extract, 0.083 g/L ferricammonium citrate, and 8.3 ml/L of a trace element solution containing 2g/L of calcium chloride dihydrate, 2.2 g/L of zinc sulfate heptahydrate,0.5 g/L manganese sulfate monohydrate, 1 g/L cuprous sulfatepentahydrate, 0.1 g/L ammonium molybdate tetrahydrate and 0.02 g/Lsodium tetraborate). The fermentor was inoculated with a lateexponential culture of E. coli W3110 containing the plasmid encoding theengineered CHMO gene of interest (grown in a shake flask as described inExample 3) to a starting OD₆₀₀ of 0.5 to 2.0. The fermentor was agitatedat 500-1500 rpm with air supplied to the fermentation vessel at 2.0-30L/min to maintain a dissolved oxygen level of 55% saturation or greater.The pH of the culture was maintained at 7.0 by addition of 28% v/vammonium hydroxide. Growth of the culture was maintained by addition ofa feed solution containing 500 g/L dextrose monohydrate, 12 g/L ammoniumchloride and 5 g/L magnesium sulfate heptahydrate. After the feed volumereaches 1000 mL expression of the CHMO polypeptide was induced byaddition of IPTG to a final concentration of 1 mM and fermentationcontinued for another 18 hours. The culture was then chilled to 4° C.and maintained at that temperature until harvested. Cells were collectedby centrifugation at 6600 G for 30 minutes at 4° C. Harvested cells wereused directly in the downstream recovery process or stored at −20 untilsuch use.

The cell pellet was resuspended in 2 volumes of 25 mM sodium phosphatebuffer, pH 7.0 at 4° C. to each volume of wet cell paste. By using 1Nsodium hydroxide pH has to be adjusted to 7.0 before lysis.Intracellular CHMO polypeptide was released from the cells by passingthe suspension through a homogenizer fitted with a two-stagehomogenizing valve assembly using a pressure of 12000 psig. The cellhomogenate was collected in chilled container which was kept in ice bathimmediately after disruption. Lysate pH has to be adjusted to 7.0 byusing 1N sodium hydroxide A solution of 11% w/v polyethyleneimine, pH7.2, was added to the lysate to a final concentration of 0.5% w/v andstirred for 30 minutes at 600 rpm at temperature of 25° C. to 30° C. Theresulting suspension was clarified by centrifugation at 6600 G in astandard laboratory centrifuge for 60 minutes at 4-8° C. The cleardecanted supernatant was cooled to 4-8° C. and pH adjusted to 7.0 byusing 1N NaOH then concentrated ten-fold using a celluloseultrafiltration membrane with a molecular weight cut off of 30 KDa at atemperature of 10° C. The final concentrate was dispensed into Petriplates, frozen at −20° C. and lyophilized for 48 h to provide the DSPpowder. The DSP powder was then transferred to polythene bags and storedat −20° C.

Assays of activity, sulfone-byproduct production, and thermal stabilityare carried out generally as described in Example 2 except that thefollowing reagents/conditions are used: 1-10 g/L DSP powder ofengineered CHMO polypeptide; 10-100 g/L pyrmetazole substrate; 1 g/LKRED coenzyme; 0.2-0.5 g/L NADP+ cofactor; 4% IPA; 96% phosphate buffer(pH 9); reaction temperature: room temperature; reaction time: 24 hr.Assay results for DSP powder preparations of engineered CHMOpolypeptides are shown below in Table 7.

TABLE 7 Thermal Stability SEQ ID NO: Activity % ee of Relative Reductionin FIOP (nt/aa) FIOP¹ S-isomer % Sulfone-byproduct³ (50° C.) 17/18 +31/32 + 96.5 35/36 + 25 51/52 ++ 57/58 ++ 1.0 30 71/72 ++ 0.6 27 75/76++ 97.8 0.2 34 85/86 ++ 99.7 0.4 40  99/100 +++ 99.8 0.2 34 107/108 +++0.1 37 123/124 ++++² (0.1%)⁴ 43 137/138 ++++² 99.8 157/158 ++++² 99.8¹Except where noted, SEQ ID NO: 18 used as “parent” for purposes ofcalculating “FIOP”. ²Assay carried out as described above with additionof 10% NMP in reaction solution. ³Values for relative reduction in %sulfone-byproduct of compound (3) are relative to the value measured forthe polypeptide of SEQ ID NO: 57 which yielded 73% conversion ofpyrmetazole substrate to esomeprazole with only 2% sulfone-byproductformation in a 24-hour reaction using 10 g/L pyrmetazole; 2.5 g/L CHMOpolypeptide DSP powder; 1 g/L KRED; 0.5 g/L NADPH; 4% v/v IPA; T =26-28° C. ⁴Value for the CHMO polypeptide SEQ ID NO: 124 is the absoluteamount of % sulfone-byproduct measured in the 100 g/L substrate loadingprocess described in Example 5. + = ≧1 fold ++ = ≧10 fold +++ = ≧50 fold++++ = ≧100 fold

Example 5 Process for Production of Esomeprazole Using Engineered CHMOPolypeptides

This example illustrates processes at two substrate loadings and aproduct workup procedure for preparing esomeprazole((S)-5-methoxy-2-((4-methoxy-3,5-dimethylpyridin-2-yl)methylsulfinyl)-1H-benzo[d]imidazoleor “compound (2b)”) in enantiomeric excess by contacting the pyrmetazolesubstrate compound (1) with an engineered CHMO polypeptide of thedisclosure (e.g., the polypeptides of SEQ ID NO: 8, 10, 12, 14, 16, 18,20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54,56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78. 80. 82, 84, 86, 88, 90,92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120,122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148,150, 152, 154, 156, 158, 210, 212, 214, 216, 218, 220, 222, 224, 226,228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254,256, 258, 260, 262, 264, or 266).

a. Exemplary Process Using 33 g/L Pyrmetazole Substrate Loading

In a 25 mL vial 360 mg of pyrmetazole were suspended in 8.52 mL ofbuffer solution (50 mM phosphate buffer at pH=9.0). Consecutively 0.48mL of IPA, 6 mg of NADP in 1 mL of buffer solution, 12 mg of KRED in 1mL of buffer solution and 12 mg of engineered CHMO polypeptide (SEQ IDNO: 100) in 1 mL of buffer solution were added. The reaction mixture wasstirred at 700 rpm in an oxygen atmosphere at room temperature (˜25° C.)with conversion periodically monitored by HPLC. After 24 h, 99%conversion of substrate to the esomeprazole product was obtained withonly 0.9% of sulfone-byproduct present. The esomeprazole product was theS-enantiomer in excess with an ee of 99.83%.

b. Exemplary Process Using 100 g/L Pyrmetazole Substrate Loading

Reaction:

In a round bottom flask with overhead stirring 7.5 g of pyrmetazolesubstrate was suspended in 47.5 mL of buffer solution (50 mM phosphatebuffer at pH=9.0) and 7.5 mL of NMP was added. The mixture was degassedand filled with oxygen 4 times prior to the addition of 3 mL of IPA.Consecutively, 15 mg of NADP in 2 mL of buffer solution (0.2 g/L), 75 mgof KRED in 10 mL of buffer solution (1 g/L) and 37.5 mg of engineeredCHMO polypeptide (SEQ ID NO: 124) in 5 mL of buffer solution (0.5 g/L)were added. The reaction mixture was a slurry that was stirred at 250rpm for 24 h at room temperature (˜25° C.) in an oxygen atmosphere. Theconversion of substrate to product was monitored periodically by HPLC.After 24 h, 23% of pyrmetazole substrate was converted to esomeprazole.Further addition of 150 mg (2 g/L) engineered CHMO polypeptide, 75 mg (1g/L) KRED and 15 mg (0.2 g/L) NADP as solids 5 times over the course ofa full reaction time of 69 h provides a final substrate conversion of99.6%.

Product Workup:

The pH of the reaction mixture was adjusted to pH=7.9 and while stirringthe slurry gets diluted with 75 mL of cold water (pH=8). The mixture wasstirred for additional 20 min at 4° C. and was filtered. The residue waswashed with additional 75 mL of ice cold water to provide a brownishfilter cake as product. The product was dried at 2-3 mbar and 40° C. toprovide 7.7 g (98% yield) of crude material. HPLC analysis indicatesthat the crude material was 97.8% pure esomeprazole with only 0.2%sulfone-byproduct of compound (3) and 1.5% pyrmetazole substrate. Theesomeprazole product was the S-enantiomer in excess with an ee of 99.9%.

Example 6 Process for Production of Esomeprazole at 30 g Scale Using anEngineered CHMO Polypeptide

This example illustrates a process for preparing esomeprazole inenantiomeric excess at a 30 g scale via a biocatalytic conversion of thesubstrate pyrmetazole using an engineered CHMO polypeptide of thedisclosure (e.g., a polypeptide of SEQ ID NO: 8, 10, 12, 14, 16, 18, 20,22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56,58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78. 80. 82, 84, 86, 88, 90, 92,94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122,124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150,152, 154, 156, or 158).

A. Biocatalytic Reaction Protocol:

A 1 L multi-neck jacketed reactor vessel equipped with baffle andoverhead stirrer, fitted with an anchor shaped agitator was charged witha pre-mixed powder of 30 g pyrmetazole (from Sinojie (HK) Ltd.) and 1.5g of an esomeprazole “seed” (prepared in a previous enzymatic reaction),and then 517 mL of 0.05 M potassium phosphate at pH 9.0 (“buffersolution”). This reaction mixture was stirred at 150 rpm at 25° C. for10 min in order to obtain a well-suspended slurry. A three way tap wasattached to the necks of the vessel. The tap was fitted with 2 rubberballoons filled with oxygen and a vacuum line for conducting degassingsteps prior to the start of the reaction. Vacuum was applied to thevessel (30 mbar, obtained within 5 min) and the evacuated flask wasfilled with oxygen via the three way tap. This evacuation-gas-fillingcycle was repeated two more times. The reaction vessel under positiveoxygen pressure then was charged sequentially with the following: 24 mLIPA (HPLC grade); 60 mg NADP in 4 mL buffer solution (pH 9.0); 300 mgketoreductase of SEQ ID NO: 268 in 15 mL buffer solution (pH 9.0); 600mg CHMO of SEQ ID NO: 158 in 40 mL buffer solution (pH 9.0); and 1.2 mLof catalase (Aspergillus niger catalase solution in buffer stabilizedwith sodium chloride and sorbitol with stated activity of 25000 CIU/g;available from Sigma-Aldrich). Catalase is added to neutralize peroxidethat may form during the reaction and negatively affect the performanceof the CHMO. The above reaction mixture was stirred at 25° C. for 48hours. The stir rate was 300 rpm in the beginning and increased stepwiseas shown in Table 8 below. The course of the reaction was followed bytaking periodic ˜0.3 mL samples from the reaction mixture which werequenched in 10 mL MeOH and analyzed using HPLC as described below. Forthe purpose of tracking the process, t=0 was set at the time at whichthe CHMO was added. Samples were also taken and tested for peroxideduring course of reaction but no peroxide was detected. The in-processreaction profile based on the sample analyzes is summarized in Table 8below. A % conversion of >99% within 36 hours can be estimated from thekinetic profile of the reaction. In comparable runs a conversion of ˜98%was determined after 32 h (with a rate of conversion of ˜1%/h constantlyat the latter stage of the reaction) and similar kinetic profile couldbe obtained in repetitive runs with the described experimental set-up.The reaction mixture 48 hours after start was taken for product work-upand isolation as described below.

TABLE 8 Reaction Profile Time Stirring speed (h) % Conversion (rpm) 0 5300 2 17.6 350 4 30.7 350 6 43.3 350 7.5 50.1 400 22.5 87.8 450 26 92.3450 28 94.3 450 30 96.1 450 44 99.6 450

B. Reaction Work-Up Protocol:

To the reaction mixture was added 165 mL (5.5 volumes) of methylisobutyl ketone (“MIBK”), the jacket temperature was adjusted to 48° C.and the mixture was stirred at 300 rpm. After 25 min the internaltemperature showed 45° C. and the slurry was completely dissolved.Mixing was stopped and after 20 min the phases were separated. The loweraqueous layer was slightly turbid with a yellowish color. The upperorganic layer appeared to be an emulsion and was brown in color. Theaqueous layer was drained and collected. The organic layer wassubsequently drained and submitted to a warm filtration over Celiteapplying vacuum (the temperature of the jacket filter was adjusted to45° C.). The aqueous phase was transferred back to the heated vessel and45 mL of MIBK was added. Stirring at 300 rpm for 30 min and phaseseparation within 20 min afforded a lower slightly turbid, yellowishaqueous layer and a brownish upper organic phase. The aqueous layer wasdrained and discarded. The organic layer was drained and collected. Thesolution was submitted to warm Celite filtration after the firstfiltration was completed (same filter and Celite layer). The organicphases were combined and separated from the aqueous layer that wasformed during filtration. The aqueous layer was discarded and theorganic phase was transferred back to the vessel. The temperature wasadjusted to 15° C. and the solution stirred at 150 rpm for 1 hour. Theproduct slowly precipitated from the solution to give a dense slurry.The temperature was further reduced to 10° C. and the slurry stirred at150 rpm for 30 minutes. Finally the temperature was adjusted to 5° C.and the mixture stirred at 150 rpm for 30 minutes. In order to increasethe mobility of the slurry 240 ml of n-heptane was added slowly in 30 mLportions (per 5 min). The slurry was drained and filtered off. Thefilter cake was dried in a vacuum oven (3-10 mm hg) at 25° C. for 72 h.A total of 28.7 g of esomeprazole was isolated (87% isolated yield) witha chemical purity of 99% by HPLC.

C. Achiral HPLC Analysis for Determination of Reaction % Conversion:

From the reaction vessel a sample of 20 μL is added into an Eppendorftube and 1.98 mL of methanol are added (100 times dilution). The sampleis centrifuged for 3 min, 100 μl of the clear supernatant solution isadded into a HPLC vial with glass insert and submitted for HPLCanalysis. Dilution is required within the linearity range if not below 1g/L. The HPLC parameters used for determination of percent conversionare shown in Table 9.

TABLE 9 Instrument Agilent HPLC 1200 series Column Waters Sunfire C18150 × 4.6 mm (5 μm), attached Waters C18 guard column Mobile phase(premixed) 53% acetonitrile, 47% water Flow rate 1.50 mL/min Detectionwavelength 250 nm Column temperature 30° C. Injection volume 10 μLRuntime 4.0 min Response factor 1.11 (at linear range 0-62.5 mg/L)(substrate area/product area) Retention Times Pyrmetazole: 3.27 minSulfone-byproduct of compound (3): 2.32 min (R)- or (S)-omeprazoleproduct: 1.99 min

D. Chiral HPLC for Determination of Chiral Analysis:

A sample of 20 μL is taken from the reaction and is evaporated to almostdryness in order to remove acetone (residual acetone is detrimental tochiral column stationary phase). The residue is dissolved in 1 mLacetonitrile (HPLC grade) and subjected to centrifugation. 100 μL of theclear supernatant solution are sampled into an HPLC vial. The solutionis diluted with 900 μL of acetonitrile and submitted for HPLC analysis.The amount of diluent should be adjusted so that the Esomeprazole peakheight maximum is maintained below 800 mAU. The chiral HPLC parametersused for determination of chiral purity are shown in Table 10.

TABLE 10 Instrument Agilent HPLC 1200 series Column Chiralpak AS-RH 150× 4.6 mm (5 μm), with AS-RH guard column Mobile phase 35% acetonitrile,65% water (premixed) Flow rate 0.6 mL/min Detection wavelength 250 nmColumn temperature 35° C. Injection volume 10 μL Runtime 30 minRetention Times (R)-omeprazole: 7.45 min (S)-omeprazole: 7.99 min

Enantioselectivity in terms of % ee was calculated based on chiral HPLCpeak areas as follows:

${\% \mspace{14mu} {ee}} = {\frac{\begin{pmatrix}{{{Peak}\mspace{14mu} {Area}\mspace{14mu} {of}\mspace{14mu} (S)\text{-}{omeprazole}} -} \\{{Peak}\mspace{14mu} {Area}\mspace{14mu} {of}\mspace{14mu} (R)\text{-}{omeprazole}}\end{pmatrix}}{\begin{bmatrix}{\left( {{Peak}\mspace{14mu} {Area}\mspace{14mu} {of}\mspace{14mu} (S)\text{-}{omeprazole}} \right) +} \\\left. {{Peak}\mspace{14mu} {Area}\mspace{14mu} (R)\text{-}{omeprazole}} \right)\end{bmatrix}} \times 100}$

Example 7 Preparation of Other Prazole Compounds Using Engineered CHMOPolypeptides

This example illustrates the use of exemplary engineered CHMOpolypeptides disclosed in Tables 2A and 2B for the conversion of sulfidesubstrate that are structurally similar to pyrmetazole to theircorresponding (R)- and/or (S)-prazole compounds that are structuralanalogs to (R)- and (S)-omeprazole. In this example, engineered CHMOpolypeptides are screened for activity and identified that are usefulfor the preparation of the prazole compounds (S)-pantoprazole,(S)-tenatoprazole, (S)-rabeprazole, and (R)- and (S)-lansoprazole, asshown in Schemes 4, 5, 6, and 7 below.

A. Preparation of S-pantoprazole

This example illustrates the use of engineered CHMO polypeptides of thepresent disclosure for carrying out the biocatalytic conversion of thesulfide precursor substrate (and pyrmetazole analog),5-(difluoromethoxy)-2-((3,4-dimethoxypyridin-2-yl)methylthio)-1H-benzo[d]imidazole,to the prazole product compound (S)-pantoprazole in enantiomeric excess,as shown in Scheme 4.

Preparation of Cell-Lysate:

A cell-lysis stock solution (1 mg/mL lysozyme, 0.5 mg/mL PMBS, 100 mMphosphate buffer, pH 10) was added 600 μA. per well to the wells of adeep-well plate each containing a cell pellet from E. coli expressingthe engineered CHMO of interest. The plate was shaken vigorously withhigh speed for 1-5 min to disperse the cell pellet then followed by1.5-2 hours of slow shaking at room temperature. The plate was thencentrifuged at 4000 rpm for 20 minutes at 4° C.

Reaction:

10 μL of the pantoprazole-precursor sulfide substrate compound(5-(difluoromethoxy)-2-((3,4-dimethoxypyridin-2-yl)methylthio)-1H-benzo[d]imidazole)stock solution (1.4 g/L in IPA) was added to each well of a deep-wellplate. Then 180 μL of a recycling system stock solution (0.2 g/L NADP+cofactor and 1 g/L KRED in 100 mM phosphate, pH 10) was also added toeach well of a deep well plate. The reaction was initiated by adding 10μL of the cell-lysate solution to each well of the deep-well plate. Theplate was sealed at 180° C. for 3.0 s, and then shaken at slow speed androom temperature for 2 h.

Analysis: 400 μL of acetonitrile was added to each well to quench thereaction. The plate was sealed again at 180° C. for 2.0 s, then shakenfor 15-20 min at room temperature, and centrifuged at 4000 rpm for 20min (25° C.). 200 μL of the supernatant of quenched solution wastransferred to the shallow well round bottom plate and this plate wassealed at 180° C. for 3 s and then shaken for 10 min. These samples inthe round bottom plate were used immediately of achiral and/or chiralHPLC analysis according to the parameters and conditions shown in Tables11 and 12 below. The samples were stored at −4° C. if the plate was notanalyzed immediately using HPLC.

TABLE 11 Achiral HPLC parameters and conditions Column Gemini-NX 5u C18,5 μm, 50 mm × 4.6 mm Solvent program 1.5 ml/min, room temperatureIsocratic at 50% ACN/50% H2O (10 mM ammonium formate) 1.3 min Injectionvolume 10 μL Detector UV-254 nm

TABLE 12 Chiral HPLC parameters and conditions Column Chiralpak IA, 5μm, 150 mm × 4.6 mm Solvent program 1.0 ml/min, 30° C. Isocratic at 40%ACN/60% H2O 7.0 min Injection volume 10 μL Detector UV-254 nm

Results:

As shown below in Table 13, the following engineered CHMO polypeptideswere capable of converting the precursor sulfide substrate to betweenabout 20 and 99 percent of the (S)-pantoprazole product in enantiomericexcess: 70, 72, 76, 78, 80, 82, 84, 86, 90, 92, 94, 96, 98, 100, 102,104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 126, 128, 130, 228,232, 244, 248, 256, 258, 260, 262, and 264. This represents betweenabout 60-fold and 310-fold improvement in percent conversion of thissubstrate to this product relative to the wild-type CHMO of SEQ ID NO:2. The engineered CHMO polypeptides of SEQ ID NO: 236 and 240 werecapable of converting the precursor sulfide substrate to 20% and 27% tothe opposite enantiomer, (R)-pantoprazole, in high enantiomeric excessesof 86 and 89%, respectively.

TABLE 13 % Conversion FIOP % ee SEQ ID NO: % Conversion (relative to SEQID NO: 2) of S-isomer 69/70 30.3 95 34.2 71/72 33.2 105 29.7 75/76 68.1215 18.7 77/78 51.1 161 41.9 79/80 39.5 124 28.1 81/82 38.5 121 27.383/84 50.6 160 49.9 85/86 96.8 306 87.6 89/90 71.8 227 82.1 91/92 68.7217 39.8 93/94 59.4 187 88.1 95/96 97.8 309 81.3 97/98 99.2 313 90.8 99/100 89.9 284 80.7 101/102 98.4 310 82.1 103/104 99.3 313 92 105/10633.9 107 95.6 107/108 98.2 310 95.2 109/110 97.5 307 95.3 111/112 99.0312 95.2 113/114 74.8 236 85.3 115/116 97.5 308 98.5 117/118 94.0 297100 119/120 99.1 313 98.9 121/122 90.8 286 99.1 125/126 98.2 310 97.4127/128 97.8 308 98.7 129/130 98.9 312 98.7 227/228 22.5 71 97.7 231/23226.7 84 85.8 235/236 20.1 63 −88.8 239/240 27.1 85 −86.9 243/244 41.9132 100 247/248 22.1 70 89.9 255/256 20.6 65 95.8 257/258 28.4 89 96.6259/260 68.2 215 92.7 261/262 46.0 145 87.5 263/264 49.3 155 91.3

B. Preparation of S-tenatoprazole

This example illustrates the use of engineered CHMO polypeptides of thepresent disclosure for carrying out the biocatalytic conversion of thesulfide precursor substrate (and pyrmetazole analog),5-methoxy-2-((4-methoxy-3,5-dimethylpyridin-2-yl)methylthio)-1H-imidazo[4,5-b]pyridine,to the prazole product compound (S)-tenatoprazole in enantiomericexcess, as shown in Scheme 5.

Preparation of Cell-Lysate:

A cell-lysis stock solution (1 mg/mL lysozyme, 0.5 mg/mL PMBS, 100 mMphosphate buffer, pH 10) was added 600 μL per well to the wells of adeep-well plate each containing a cell pellet from E. coli expressing aCHMO of interest. The plate was shaken vigorously with high speed for1-5 min to disperse the cell pellet then followed by 1.5-2 hours of slowshaking at room temperature. The plate was then centrifuged at 4000 rpmfor 20 minutes at 4° C.

Reaction:

10 μL of the tenatoprazole-precursor sulfide substrate compound(5-methoxy-2-((4-methoxy-3,5-dimethylpyridin-2-yl)methylthio)-1H-imidazo[4,5-b]pyridine)stock solution (0.65 g/L in IPA) was added to each well of a deep-wellplate. Then 180 μL of a recycling system stock solution (0.2 g/L NADP+cofactor and 1 g/L KRED in 100 mM phosphate, pH 10) was also added toeach well of a deep well plate. The reaction was initiated by adding 10μL of the cell-lysate solution to each well of the deep-well plate. Theplate was sealed at 180° C. for 3.0 s, and then shaken at slow speed androom temperature for 2 h.

Analysis:

400 μL of acetonitrile was added to each well to quench the reaction.The plate was sealed again at 180° C. for 2.0 s, then shaken for 15-20min at room temperature, and centrifuged at 4000 rpm for 20 min (25°C.). For achiral HPLC sample preparation, 200 μL of the supernatant ofquenched solution was transferred to the shallow-well round bottom plateand this plate was sealed at 180° C. for 3 s and then shaken for 10 min.These samples in the round bottom plate were used immediately forachiral HPLC analysis according to the parameters and conditions shownin Table 14 below. The samples were stored at −4° C. if the plate wasnot analyzed immediately using HPLC.

TABLE 14 Achiral HPLC parameters and conditions (for determining %conversion) Column Gemini-NX 5u C18, 5 μm, 50 mm × 4.6 mm Solventprogram 1.5 ml/min, 30° C. Isocratic at 50% ACN/50% H2O (10 mM ammoniumformate) 1.5 min Injection volume 10 μL Detector UV-254 nm

For chiral HPLC sample preparation, the 200 μL of the supernatant ofquenched solution in the shallow-well round bottom plates was fullyevaporated in a vacuum oven and 200 μL of MTBE was added to each well.The plate was sealed at 180° C. for 2 sec and shaken for 10 min thenused immediately for chiral HPLC analysis according to the parametersand conditions shown in Table 15 below. The samples were stored at −4°C. if the plate was not analyzed immediately using HPLC.

TABLE 15 Chiral HPLC parameters and conditions Column Chiralpak AS-H, 5μm, 250 mm × 4.6 mm Solvent program 0.6 ml/min, 20° C. Isocratic at 20%hexane/80% ethanol 11.0 min Injection volume 5 μL Detector UV-254 nm

Results:

As shown below in Table 16, the following engineered CHMO polypeptideswere capable of converting the precursor sulfide substrate to betweenabout 10 and 60 percent of the (S)-tenatoprazole product in 100% ee: 66,102, 104, 114, 122, 124, 128, 232, 238, 244, 260, 262, and 264. Thisrepresents between about 10-fold and 60-fold improvement in percentconversion of this substrate to this product relative to the wild-typeCHMO of SEQ ID NO: 2.

TABLE 16 % Conversion FIOP % ee SEQ ID NO: % Conversion (relative to SEQID NO: 2) of S-isomer 65/66 13.3 22.9 100 101/102 21.1 36.3 100 103/10425.8 44.4 100 113/114 16.3 27.9 100 121/122 17.6 30.2 100 123/124 6.811.7 100 127/128 9.1 15.6 100 231/232 33.4 57.3 100 237/238 11.8 20.3100 243/244 30.9 53.0 100 259/260 35.9 61.7 100 261/262 30.4 52.1 100263/264 29.0 49.9 100

C. Preparation of S-rabeprazole

This example illustrates the use of engineered CHMO polypeptides of thepresent disclosure for carrying out the biocatalytic conversion of thesulfide precursor substrate (and pyrmetazole analog),2-((4-(3-methoxypropoxy)-3-methylpyridin-2-yl)methylthio)-1H-benzo[d]imidazole,to the prazole compound (S)-rabeprazole in enantiomeric excess, as shownin Scheme 6.

Preparation of Cell-Lysate:

A cell-lysis stock solution (1 mg/mL lysozyme, 0.5 mg/mL PMBS, 100 mMphosphate buffer, pH 10) was added 600 μL per well to the wells of adeep-well plate each containing a cell pellet from E. coli expressingthe engineered CHMO of interest. The plate was shaken vigorously withhigh speed for 1-5 min to disperse the cell pellet then followed by1.5-2 hours of slow shaking at room temperature. The plate was thencentrifuged at 4000 rpm for 20 minutes at 4° C.

Reaction:

10 μL of the rabeprazole-precursor sulfide substrate compound(2-((4-(3-methoxypropoxy)-3-methylpyridin-2-yl)methylthio)-1H-benzo[d]imidazole)stock solution (1.9 g/L in IPA) was added to each well of a deep-wellplate. Then 180 μL of a recycling system stock solution (0.2 g/L NADP+cofactor and 1 g/L KRED in 100 mM phosphate, pH 10) was also added toeach well of a deep well plate. The reaction was initiated by adding 10μL of the cell-lysate solution to each well of the deep-well plate. Theplate was sealed at 180° C. for 3.0 s, and then shaken at slow speed androom temperature for 2 h.

Analysis:

400 μL of acetonitrile was added to each well to quench the reaction.The plate was sealed again at 180° C. for 2.0 s, then shaken for 15-20min at room temperature, and centrifuged at 4000 rpm for 20 min (25°C.). For achiral HPLC sample preparation, 200 μL of the supernatant ofquenched solution was transferred to the shallow-well round bottom plateand this plate was sealed at 180° C. for 3 s and then shaken for 10 min.These samples in the round bottom plate were used immediately forachiral HPLC analysis according to the parameters and conditions shownin Table 17 below. The samples were stored at −4° C. if the plate wasnot analyzed immediately using HPLC.

TABLE 17 Achiral HPLC parameters and conditions Column Gemini-NX 5u C18,5 μm, 50 mm × 4.6 mm Solvent program 1.5 mL/min, 30° C. Isocratic at 50%ACN/50% H₂O (10 mM ammonium formate) 1.5 min Injection volume 10 μLDetector UV-254 nm

For chiral HPLC sample preparation, the 200 μL of the supernatant ofquenched solution in the shallow-well round bottom plates was fullyevaporated in a vacuum oven and 200 μL of MTBE was added to each well.The plate was sealed at 180° C. for 2 sec and shaken for 10 min thenused immediately for chiral HPLC analysis according to the parametersand conditions shown in Table 18 below. The samples were stored at −4°C. if the plate was not analyzed immediately using HPLC.

TABLE 18 Chiral HPLC parameters and conditions Column Chiralpak AD-H, 5μm, 250 mm × 4.6 mm Solvent program 1.0 mL/min, 30° C. Isocratic at 65%hexane/20% ethanol/ 15% isopropyl alcohol 10.0 min Injection volume 5 μLDetector UV-254 nm

Results:

Use of the wild-type CHMO of SEQ ID NO: 2 did not result in anydetectable conversion of2-((4-(3-methoxypropoxy)-3-methylpyridin-2-yl)methylthio)-1H-benzo[d]imidazoleto (R)- or (S)-rabeprazole. As shown below in Table 19, the followingengineered CRMO polypeptides were capable of converting the precursorsulfide substrate to between about 1 and 10 percent of the(S)-rabeprazole product: 62, 76, 84, 86, 124, 238, 240, 250, 258, 262,and 264. Chiral HPLC analysis of the two polypeptides with the highest %conversion, SEQ ID NO: 76 and 86, showed that they produced the(S)-rabeprazole product in 100% ee.

TABLE 19 % ee SEQ ID NO: % Conversion of S-isomer 61/62 1.56 75/76 3.25100 83/84 2.19 85/86 10.27 100 123/124 1.49 237/238 1.89 239/240 1.20249/250 1.81 257/258 3.06 261/262 3.06 263/264 2.43

D. Preparation of S-lansoprazole or R-lansoprazole (“Dexlansoprazole”)

This example illustrates the use of engineered CHMO polypeptides of thepresent disclosure for carrying out the biocatalytic conversion of thesulfide precursor substrate (and pynnetazole analog),2-((3-methyl-4-(2,2,2-trifluoroethoxy)pyridin-2-yl)methylthio)-1H-benzo[d]imidazole,to either of the prazole compounds (S)-lansoprazole or (R)-lansoprazolein enantiomeric excess, as shown in Scheme 7.

Screening assays using the sulfide precursor substrate2-((3-methyl-4-(2,2,2-trifluoroethoxy)pyridin-2-yl)methylthio)-1H-benzo[d]imidazolewere carried out with certain engineered CHMO polypeptides of thepresent disclosure. Assay conditions and results for % conversion andproduct enantioselectivity are provided below in Table 20.

TABLE 20 Enantioselectivity SEQ ID NO: % Conversion (% ee) 5/6  1.2¹R-selective (n.d.)⁴ 21/22  1.1² S-selective (n.d.)⁴ 71/72 27³S-selective (71.7% ee) 79/80 19³ S-selective (91.6% ee) ¹1.5 g/Llansoprazole sulfide, 15 g/L enzyme, 0.5 g/L NADP⁺, 1 g/L KRED, 4% IPA,25 mM phosphate buffer pH 8.5, 17 h reaction time. ²2.0 g/L lansoprazolesulfide, 25 g/L enzyme, 0.5 g/L NADP⁺, 1 g/L KRED, 4% IPA, 25 mMphosphate buffer pH 8.5, 17 h reaction time. ³1.5 g/L lansoprazolesulfide, 5 g/L enzyme, 0.5 g/L NADP+, 1 g/L KRED, 4% IPA, 25 mMphosphate buffer pH 8.5, 24 h reaction time. ⁴% ee could not bedetermined due to low % conversion of substrate to product.

As shown in Table 20, the engineered CHMO polypeptides of SEQ ID NO: 22,72, and 80, are capable of converting the lansoprazole sulfide precursorsubstrate to (S)-lansoprazole in enantiomeric excess. The polypeptidesof SEQ ID NO: 72 and 80 are capable of 27% and 19% conversion withenantioselectivity of about 72% ee and 92% ee, respectively. Althoughthe engineered CHMO polypeptide of SEQ ID NO: 6 showed only 1%conversion it was confirmed to be selective for the (R)-lansoprazoleproduct. The polypeptide of SEQ ID NO: 6 was also found to convertpyrmetazole to favor the (R)-omeprazole product over the (S)-omeprazoleproduct in 98.9% enantiomeric excess (see Table 2A). It is reasonable toexpect that further directed evolution of the engineered polypeptide ofSEQ ID NO: 6 will result in an engineered CHMO polypeptide capable ofproducing the (R)-lansoprazole product in high enantiomeric excess(e.g., 98% or greater) and much higher % conversion (e.g., 20% orgreater).

All publications, patents, patent applications and other documents citedin this application are hereby incorporated by reference in theirentireties for all purposes to the same extent as if each individualpublication, patent, patent application or other document wereindividually indicated to be incorporated by reference for all purposes.

While various specific embodiments have been illustrated and described,it will be appreciated that various changes can be made withoutdeparting from the spirit and scope of the invention(s).

1. A non-naturally occurring monooxygenase comprising a polypeptidecapable of converting5-methoxy-2-((4-methoxy-3,5-dimethylpyridin-2-yl)methylthio)-1H-benzo[d]imidazole(“compound (1)”) to5-methoxy-2-((4-methoxy-3,5-dimethylpyridin-2-yl)methylsulfinyl)-1H-benzo[d]imidazole(“compound (2)”), and which polypeptide amino acid sequence: (a) is atleast 80% identical to SEQ ID NO: 124; and (b) has two or more residuedifferences as compared to SEQ ID NO:2 at the following residuepositions: position corresponding to X246; position corresponding toposition corresponding to X248; position corresponding to X326; positioncorresponding to X386; position corresponding to X432; positioncorresponding to X433; position corresponding to X435; positioncorresponding to X438; and position corresponding to X448.
 2. (canceled)3. The non-naturally occurring monooxygenase of claim 1 in which thepolypeptide is capable of converting the compound (1) to(S)-5-methoxy-2-((4-methoxy-3,5-dimethylpyridin-2-yl)methylsulfinyl)-1H-benzo[d]imidazole(“compound (2b)”) in enantiomeric excess.
 4. The non-naturally occurringmonooxygenase of claim 3 in which the amino acid sequence comprises atleast comprises an amino acid sequence in which at least residue atposition corresponding to X326 is C.
 5. The non-naturally occurringmonooxygenase of claim 3 in which the amino acid sequence comprises atleast comprises an amino acid sequence in which at least residue atposition corresponding to X386 is S.
 6. The non-naturally occurringmonooxygenase of claim 3 in which the amino acid sequence comprises atleast the following features: residue at position corresponding to X326is C; residue at position corresponding to X386 is S; residue atposition corresponding to X432 is A, or L.
 7. The non-naturallyoccurring monooxygenase of claim 6 in which the amino acid sequencefurther comprises one or more of the following features: residue atposition corresponding to X433 is A, L, or V; residue at positioncorresponding to X435 is S; residue at position corresponding to X438 isI; residue at position corresponding to X448 is V or W; and residue atposition corresponding to X490 is R.
 8. (canceled)
 9. (canceled)
 10. Thenon-naturally occurring monooxygenase of claim 3 in which the amino acidsequence comprises one or more of the following features: residue atposition corresponding to X3 is T; residue at position corresponding toX14 is A; residue at position corresponding to X34 is K; residue atposition corresponding to X43 is G; residue at position corresponding toX71 is M; residue at position corresponding to X83 is E; residue atposition corresponding to X111 is T; residue at position correspondingto X141 is I or V; residue at position corresponding to X174 is L or I;residue at position corresponding to X209 is P; residue at positioncorresponding to X240 is F, or K; residue at position corresponding toX246 is E, H, Q, S, R, W, or Y; residue at position corresponding toX248 is C, I, N, V, or S; residue at position corresponding to X277 isL, M, or Q; residue at position corresponding to X278 is G, N, or S;residue at position corresponding to X280 is G, or R; residue atposition corresponding to X281 is A, or S; residue at positioncorresponding to X282 is S; residue at position corresponding to X288 isI; residue at position corresponding to X307 is C or R; residue atposition corresponding to X341 is E; residue at position correspondingto X373 is L; residue at position corresponding to X377 is G; residue atposition corresponding to X388 is K; residue at position correspondingto X390 is R or I; residue at position corresponding to X400 is I;residue at position corresponding to X415 is A; residue at positioncorresponding to X426 is F; residue at position corresponding to X449 isM, F or L; residue at position corresponding to X464 is D; residue atposition corresponding to X481 is K; residue at position correspondingto X488 is F, K, or L; residue at position corresponding to X489 is a C;residue at position corresponding to X499 is L; residue at positioncorresponding to X505 is W or L; residue at position corresponding toX516 is V; residue at position corresponding to X526 is V; residue atposition corresponding to X537 is T; and residue at positioncorresponding to X540 is Q, or A.
 11. The non-naturally occurringmonooxygenase of claim 3 in which the amino acid sequence comprises asequence selected from the group consisting of SEQ ID NO: 8, 10, 12, 14,16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50,52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76,
 78. 80. 82, 84, 86,88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116,118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144,146, 148, 150, 152, 154, 156, 158, 210, 212, 214, 216, 218, 220, 222,224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250,252, 254, 256, 258, 260, 262, 264, and
 266. 12. The non-naturallyoccurring monooxygenase of claim 3 in which the amino acid sequencecomprises at least the following features: (a) residue at positioncorresponding to X326 is C; residue at position corresponding to X386 isS; residue at position corresponding to X432 is A, or L; residue atposition corresponding to X433 is A; residue at position correspondingto X435 is S; and residue at position corresponding to X490 is R; (b)residue at position corresponding to X34 is K; residue at positioncorresponding to X209 is P; residue at position corresponding to X240 isK; residue at position corresponding to X288 is I; residue at positioncorresponding to X326 is C; residue at position corresponding to X386 isS; residue at position corresponding to X415 is A; residue at positioncorresponding to X432 is L; residue at position corresponding to X433 isA; residue at position corresponding to X435 is S; residue at positioncorresponding to X438 is I; residue at position corresponding to X448 isV; residue at position corresponding to X490 is R; residue at positioncorresponding to X516 is V; and residue at position corresponding toX537 is T; (c) residue at position corresponding to X14 is A; residue atposition corresponding to X34 is K; residue at position corresponding toX141 is V; residue at position corresponding to X209 is P; residue atposition corresponding to X240 is K; residue at position correspondingto X288 is I; residue at position corresponding to X326 is C; residue atposition corresponding to X386 is S; residue at position correspondingto X415 is A; residue at position corresponding to X426 is F; residue atposition corresponding to X432 is L; residue at position correspondingto X433 is A; residue at position corresponding to X435 is S; residue atposition corresponding to X438 is I; residue at position correspondingto X448 is V; residue at position corresponding to X489 is C; residue atposition corresponding to X490 is R; residue at position correspondingto X516 is V; and residue at position corresponding to X537 is T; (d)residue at position corresponding to X14 is A; residue at positioncorresponding to X34 is K; residue at position corresponding to X111 isT; residue at position corresponding to X141 is V; residue at positioncorresponding to X209 is P; residue at position corresponding to X240 isK; residue at position corresponding to X246 is Y; residue at positioncorresponding to X288 is I; residue at position corresponding to X307 isR; residue at position corresponding to X326 is C; residue at positioncorresponding to X386 is S; residue at position corresponding to X388 isK; residue at position corresponding to X415 is A; residue at positioncorresponding to X426 is F; residue at position corresponding to X432 isL; residue at position corresponding to X433 is A; residue at positioncorresponding to X435 is S; residue at position corresponding to X438 isI; residue at position corresponding to X448 is V; residue at positioncorresponding to X481 is K; residue at position corresponding to X489 isC; residue at position corresponding to X490 is R; residue at positioncorresponding to X505 is L; residue at position corresponding to X516 isV; and residue at position corresponding to X537 is T; (e) residue atposition corresponding to X14 is A; residue at position corresponding toX34 is K; residue at position corresponding to X43 is G; residue atposition corresponding to X71 is M; residue at position corresponding toX111 is T; residue at position corresponding to X141 is V; residue atposition corresponding to X149 is W; residue at position correspondingto X209 is P; residue at position corresponding to X240 is K; residue atposition corresponding to X246 is Y; residue at position correspondingto X248 is V; residue at position corresponding to X277 is M; residue atposition corresponding to X288 is I; residue at position correspondingto X307 is R; residue at position corresponding to X326 is C; residue atposition corresponding to X386 is S; residue at position correspondingto X388 is K; residue at position corresponding to X390 is I; residue atposition corresponding to X415 is A; residue at position correspondingto X426 is F; residue at position corresponding to X432 is L; residue atposition corresponding to X433 is A; residue at position correspondingto X435 is S; residue at position corresponding to X438 is I; residue atposition corresponding to X448 is V; residue at position correspondingto X481 is K; residue at position corresponding to X489 is C; residue atposition corresponding to X490 is R; residue at position correspondingto X499 is L; residue at position corresponding to X505 is L; residue atposition corresponding to X516 is V; residue at position correspondingto X526 is V; residue at position corresponding to X537 is T; andresidue at position corresponding to X540 is Q; or (g) residue atposition corresponding to X14 is A; residue at position corresponding toX34 is K; residue at position corresponding to X43 is G; residue atposition corresponding to X71 is M; residue at position corresponding toX83 is E; residue at position corresponding to X111 is T; residue atposition corresponding to X141 is V; residue at position correspondingto X149 is W; residue at position corresponding to X174 is I; residue atposition corresponding to X209 is P; residue at position correspondingto X240 is F or K; residue at position corresponding to X246 is Y;residue at position corresponding to X248 is I or V; residue at positioncorresponding to X277 is M; residue at position corresponding to X288 isI; residue at position corresponding to X307 is C or R; residue atposition corresponding to X326 is C; residue at position correspondingto X341 is E; residue at position corresponding to X373 is L; residue atposition corresponding to X377 is G; residue at position correspondingto X383 is G; residue at position corresponding to X386 is S; residue atposition corresponding to X388 is K; residue at position correspondingto X390 is I; residue at position corresponding to X400 is I; residue atposition corresponding to X415 is A; residue at position correspondingto X426 is F; residue at position corresponding to X432 is L; residue atposition corresponding to X433 is A; residue at position correspondingto X435 is S; residue at position corresponding to X438 is I; residue atposition corresponding to X448 is V; residue at position correspondingto X449 is F; residue at position corresponding to X464 is D; residue atposition corresponding to X481 is K; residue at position correspondingto X488 is K; residue at position corresponding to X489 is C; residue atposition corresponding to X490 is R; residue at position correspondingto X499 is L; residue at position corresponding to X505 is L; residue atposition corresponding to X516 is V; residue at position correspondingto X526 is V; residue at position corresponding to X537 is T; andresidue at position corresponding to X540 is Q.
 13. The non-naturallyoccurring monooxygenase of claim 1 in which the polypeptide is capableof converting the compound (1) to(R)-5-methoxy-2-(4-methoxy-3,5-dimethylpyridin-2-yl)methylsulfinyl)-1H-benzo[d]imidazole(“compound (2a)”) in enantiomeric excess, and in which the amino acidsequence comprises at least two or more of the following features:residue at position corresponding to X432 is A, C, D, H, K, I, S, T, orY; residue at position corresponding to X433 is A, F, G, K, or W;residue at position corresponding to X435 is A or S; and residue atposition corresponding to X490 is R.
 14. (canceled)
 15. Thenon-naturally occurring monooxygenase of claim 13 in which the aminoacid sequence comprises a sequence selected from the group consisting ofSEQ ID NO: 4, 6, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180,182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, and208.
 16. (canceled)
 17. A polynucleotide encoding the polypeptide ofclaim
 1. 18. (canceled)
 19. (canceled)
 20. (canceled)
 21. (canceled) 22.(canceled)
 23. A process for converting a substrate compound ofstructural formula (I) to the product compound of formula (II):

wherein, Ar¹ is an optionally substituted aryl or heteroaryl ring; R isH, a lower alkyl, a heteroalkyl, or forms a 5 to 8 membered cycloalkyl,heteroalkyl, aryl or heteroaryl fused ring with a ring carbon of Ar¹;and Ar² is an optionally substituted cycloalkyl, heterocycloalkyl, aryl,or heteroaryl ring fused to the imidazole ring; the process comprising:contacting the compound of formula (I) with a polypeptide of claim 1 inpresence of a cofactor under suitable reaction conditions for convertingthe substrate to the product compound of formula (II).
 24. The processof claim 23 in which Ar¹ is an optionally substituted phenyl or pyridyl.25. The process of claim 23 in which Ar² is an optionally substitutedthienyl, phenyl or pyridyl.
 26. (canceled)
 27. (canceled)
 28. Theprocess of claim 23 in which the compound of formula (I) is compound (1)and the product compound of formula (II) is compound (2a), wherein thecompound (2a) is formed in enantiomeric excess.
 29. The process of claim28 in which the polypeptide comprises an amino acid sequence having theset of amino acid differences relative to SEQ ID NO: 2 that are presentin any one of SEQ ID NO: 4, 6, 160, 162, 164, 166, 168, 170, 172, 174,176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202,204, 206, and
 208. 30. (canceled)
 31. The process of claim 23 in whichthe compound of formula (I) is compound (1) and the product compound offormula (II) is compound (2b), wherein the compound (2b) is formed inenantiomeric excess.
 32. The process of claim 31 in which thepolypeptide comprises an amino acid sequence having the set of aminoacid differences relative to SEQ ID NO: 2 that are present in any one ofSEQ ID NO: 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36,38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72,74, 76,
 78. 80. 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106,108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134,136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 210, 212,214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240,242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, and
 266. 33.(canceled)
 34. (canceled)
 35. (canceled)
 36. (canceled)
 37. The processof claim 23 in which the compound of formula (II) is an omeprazoleanalog compound selected from: (R) or (S)-lansoprazole, (R) or(S)-tenatoprazole, (R) or (S)-rabeprazole, (R) or (S)-pantoprazole, (R)or (S)-ilaprazole, (R) or (S)-leminoprazole, (R) or (S)-saviprazole, and(R) or (S)-TY-11345, and the compound of formula (I) is thecorresponding pyrmetazole analog compound.
 38. (canceled)
 39. (canceled)40. (canceled)
 41. (canceled)
 42. (canceled)
 43. (canceled) 44.(canceled)
 45. (canceled)
 46. (canceled)
 47. (canceled)
 48. (canceled)49. (canceled)
 50. (canceled)
 51. (canceled)
 52. The process of claim 23in which the reaction condition comprises a partial pressure of O₂ atgreater than atmospheric pressure.
 53. (canceled)
 54. (canceled) 55.(canceled)
 56. (canceled)
 57. (canceled)
 58. (canceled)
 59. (canceled)